OA12899A

OA12899A - Synergistic combination of an alphan-2-delta ligand and a PDEV inhibitor for use in the treatment ofpain.

Info

Publication number: OA12899A
Application number: OA1200500037A
Authority: OA
Inventors: Mark John Field; Richard Griffith Williams
Original assignee: Pfizer
Priority date: 2002-08-15
Filing date: 2003-08-04
Publication date: 2006-10-13
Also published as: CA2495433A1; NZ537818A; UY27936A1; NO20050782L; IS7643A; UA77329C2; EA007504B1; PA8577301A1; MA27381A1; RS20050078A; AR040871A1; AU2003249476B2; MXPA05001835A; SI1536782T1; GEP20084366B; EA200500207A1; HRP20050136A2; JP2006502139A; CN1674884A; AP2005003227A0

Abstract

The instant invention relates to a combination of an alpha-2-delta ligand and a PDEV inhibitor for use in therapy, particularly in the curative, prophylactic or palliative treatment of pain, particularly neuropathic pain. Particularly preferred alpha-2-delta ligands are gabapentin and pregabalin. Particularly preferred PDEV inhibitors are sildenafil, vardenafil and tadalafil.

Description

012899

SYNERGISTIC COMBINATION QF AN ALPHA-2-DELTA LIGAND AND A PDEV

INHIBITQR FOR USE IN THE TREATMENT QF PAIN

HELP QF THE INVENTION

5 This invention relates to combinations of an alpha-2-delta ligand and a cGMP PDEV (‘PDEV’) inhibitor, particularly those which exhibit a synergistic effect,particularly for the curative, prophylactic or palliative treatment of pain and relateddisorders. 10 15 20

25

BACKGROUND TO THE INVENTION

Alpha-2-delta ligands may be defined as compounds which selectivelydisplace 3H-gabapentin from porcine brain membranes indicating a high affinityinteraction with the alpha-2-delta (α2δ) subunit of voltage-gated calcium channels.Alpha-2-delta ligands also includes compounds which do not displace 3H-gabapentin,but are structurally similar to compounds that do, which might be expected to bindalpha-2-delta at a slightîy different site than 3H-gabapentin or may bind to humanbrain alpha-2-delta but not porcine alpha-2-delta. They may also be known as G AB Aanalogs.

Alpha-2-delta ligands hâve been described for a number of indications. Thebest known alpha-2-delta ligand, gabapentin (NEURONTIN®), l-(aminomethyl)-cyclohexylacetic acid, was first described in the patent literature in the patent familycomprising US4024175. The compound is approved for the treatment of epilepsy andneuropathie pain. A second alpha-2-delta ligand, pregabalin, (S)-(+)-4-amino-3-(2~methylpropyl)butanoic acid, is described in European patent application publicationnumber EP641330 as an anti-convulsant treatment useful in the treatment of epilepsy 30 and in EP0934061 for the treatment of pain. 012899

Further WO0128978, describes a sériés of alpha-2-delta ligands, particularly(la,3a,5a)(3-amino-methyl-bicyclo[3.2.0]hept-3-yl)-acetic acid, depicted below: NH2 ço2h

More recently, International Patent Application Number PCT/IB02/01146(unpublished at the priority date of the présent invention) and published asWO02/085839, describes a sériés of alpha-2-delta ligands of the following formulae: HO,C NH, HO,C NH, HOaC NH. HO2C NH,

L J R1 —f”"R2

Ri—1.....f-R2 (II) R1^R2 R1 02 (III) (IV) HO,Ç NH, HO,Ç NH, HO,Ç NH, HO,Ç N H, NH, HO^ R2

(VI) R1 R2 (V) R2 (VU)

(VIH) R1 R2

H,N HO,C. k Halk Η,Ν^ Η,Ν^

ho2c^„..|-0 HO,C^jQ HD2C^jQ HO,C NH, (IX) (X) (XI) (XII) HO2C i>ih, H02ç nh2 H02ç IjlH,R1 h„>X,,.R2 HO,C NH, (XIII) (XIV) H,N.

H,N

HO.C S..·

HO.C.

XVIII

XIX HO.C f R1 R2(XV) H,N.

Ri R2(XVI) Λ R1 R2(XVII) H,N.

HO.C H,N. HO.C.

HO,C. H,N.

HO,C

XX

XXI

H.N Η2Νχ

XXII

XXIII

XXIV

XXV wherein R1 and R2 are each independently selected from H, straight or branched alkyl of 1-6 carbon atoms, cycloalkyl of from 3-6 carbon atoms, phenyl and benzyl, subject to the proviso that, except in the case of a tricyclooctane compound of formula (XVH), R1 and R2 are not simultaneously hydrogen; for use in the treatment 5 of a number of indications, including pain.

International Patent application No. PCT/IB03/00976, unpublished at thefiling date of the présent invention, describes compounds of the formula I, below:

10 wherein Ri is hydrogen or (Ci-Ce)alkyl optionally substituted with from one to five fluorine atoms; R2 is hydrogen or (Ci-Cé)alkyl optionally substituted with from one to fivefluorine atoms; or

Ri and R2, together with the carbon to which they are attached, form a three to 15 six membered cycloalkyl ring; R3 is (C]-C6)alkyl, (C3-C6)cycloalkyl, (C3-C6)cycloalkyl-(Ci-C3)alkyl, phenyl,phenyl-(Ci-C3)alkyl, pyridyl, pyridyl-(Ci-C3)alkyl, phenyl-N(H)-, or pyridyl-N(H)- , 1 wherein each of the foregoing alkyl moieties can be optionally substituted with fromone to five fluorine atoms, preferably with from zéro to three fluorine atoms, and 20 wherein said phenyl and said pyridyl and the phenyl and pyridyl moieties of saidphenyl-(Ci-C3)alkyl and said pyridyl-(Ci-C3)alkyl, respectively, can be optionallysubstituted with from one to three substituents, preferably with from zéro to twosubstituents, independently selected from chloro, fluoro, amino, nitro, cyano, (C3-C3)alkylamino, (Ci-C3)alkyl optionally substituted with from one to three fluorine 25 atoms and (C]-C3)alkoxy optionally substituted with from one to three fluorine atoms; R4 is hydrogen or (Ci~C6)alkyI optionally substituted with from one to five fluorine atoms; R5 is hydrogen or (Ci-Cg)alkyl optionally substituted with from one to fivefluorine atoms; and --—Ro is hydrogcn or (Ci G-^aik-y-1;---- and the pharmaceutically acceptable salts of such compounds.

Inhibitors of the cyclic guanosine 3’, 5’-monophosphate phosphodiesterase * 5 type five (cGMP PDEV) enzyme (‘PDEV inhibitors’) may be characterized bycompounds with high affinity and selectivity for the PDEV enzyme with little or noaffinity for the other phosphodiesterase isoforms and they hâve been described asbeing useful for treating a number of indications. In particular, sildenafil (5-[2-ethoxy-5-(4-methyl-l-piperazinylsulphonyl)phenyl]-l-methyl-3-n-propyl-l,β- 10 dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one) (VIAGRA®) has been described for the V )treatment of a number of cardiovascular disorders and has proved to be successful asthe first orally effective treatment for male erectile dysfunction (MED). The use ofPDEV inhibitors in the treatment of neuropathy has been described in EuropeanPatent Application Number EPI 129706 and WO01Z26659. Analgésie effects of 15 sildenafil hâve recently been described in Jain et al, Brain Research, 909, 170-178(2001); Asomoza-Espinosa et al, Eur. J. Pharm., 418, 195-200 (2001); and Mixcoatl-Zecutal et al, Eur. J. Pharm., 400, 81-87 (2001).

SUMMARY OF THE INVENTION 20

It has now been found that combination therapy with an alpha-2-delta ligandand a PDEV inhibitor results in unexpected improvement in the treatment of pain.

When administered simultaneously, sequentially or separately, the alpha-2-deltaligand and PDEV inhibitor interact in a synergistic manner to control pain. This 25 unexpected synergy allows a réduction in the dose required of each compound,leading to a réduction in the side effects and enhancement of the clinical effectivenessof the compounds and treatment.

Accordingly, as a first aspect, the invention provides a combination product 30 comprising an alpha-2-delta ligand, excluding pregabalin and gabapentin, and aPDEV inhibitor. Alternative! y, the exclusion may also include the compounds (i)-(xxv) of PCTÆB02/01146. 012899

As an alternative or further aspect, the invention provides a synergisticcombination product comprising an alpha-2-delta ligand and a PDEV inhibitor. 5 Examples of alpha-2-delta ligands for use with the présent invention are those compounds generally or specifically disclosed in US4024175, particularly gabapentin,EP641330, particularly pregabalin, US5563175, WO9733858, WO9733859,W09931057, WO9931074, WO9729101, WO02085839, particularly [(lR,5R,6S)-6-(Aminomethyl)bicyclo[3.2.0]hept-6-yl]acetic acid, WO9931075, particularly 3-(l- 10 Aminomethyl-cyclohexylmethyl)- 4H-[l,2,4]oxadiazol-5-one and C-[l-(lH-Tetrazol-5-ylmethyl)-cycloheptyl]- methylamine, WO9921824, particularly (3S,4S)-(l-Aminomethyl-3,4-dimethyl-cyclopentyl)-acetic acid, W00190052, WO0128978, particularly (1α,3α,5α)(3-amino-methyl-bicyclo[3.2.0]hept-3-yl)-acetic acid, EP0641330, WO9817627, 15 W00076958, particularly (3S,5R)-3-aminomethyl-5-methyl-octanoic acid, PCT/IB03/00976, particularly (3S,5R)-3-amino-5-methyl-heptanoic acid, (3S,5R)-3-amino-5-methyl-nonanoic acid and (3S,5R)-3-Amino-5-methyl-octanoic acid,EP1178034, EP1201240, WO9931074, W003000642, WO0222568, W00230871,W00230881, W002100392, W002100347, WO0242414, WO0232736 and 20 WO0228881, and pharmaceutically acceptable salts and solvatés thereof, ail of which are incorporated herein by reference.

Preferred alpha-2-delta ligands of the présent invention include: gabapentin,pregabalin, [(lR,5R,6S)-6-(Aminomethyl)bicyclo[3.2.0]hept-6-yl]acetic acid, 3-(l- 25 Aminomethyl-cyclohexylmethyl)-4H-[l,2,4]oxadiazol-5-one and C-[1-(1H-Tetrazol- 5-ylmethyl)-cycloheptyl]-methylamine, (3S,4S)-(l-Aminomethyl-3,4-dimethyl-cyclopentyl)-acetic acid, (la,3o',5a)(3-amino-methyl-bicyclo[3.2.0]hept-3-yl)-aceticacid, (3S,5R)-3-Aminomethyl-5-methyl-octanoic acid, (3S,5R)-3-amino-5-methyl-heptanoic acid, (3S,5R)-3-amino-5-methyl-nonanoic acid and (3S,5R)-3-Amino- 30 5-methyl-octanoic acid. 012899

UsefuI cyclic alpha-2-delta ligands of the présent invention may be depictedby the following formula (T):

5 wherein X is a carboxylic acid or carboxylic acid bioisostere;n is 0, 1 or 2; and R1, Rla, R2, R2a, R3, R3a, R4 and R4a are independently selected from H and Cj-Csalkyl, or R1 and R2 or R2 and R3 are taken together to form a C3-C7 cycloalkyl ring, which is10 optionally substituted with one or two substituents selected from Ci-Cg alkyl, or a pharmaceutically acceptable sait or solvaté thereof.

In formula (I), suitably, R1, Rla, R2a, R3a, R4 and R4a are H and R2 and R3 areindependently selected from H and methyl, or Rla, R2a, R3a and R4a are H and R1 and 15 R2 or R2 and R3 are taken together to form a C3-C7 cycloalkyl ring, which isoptionally substituted with one or two methyl substituents. A suitable carboxylic acidbioisostere is selected from tetrazolyl and oxadiazolonyl. X is preferably a carboxylicacid. 20 In formula (I), preferably, R1, Rla, R211, R3a, R4 and R4a are H and R2 and R3 are independently selected from H and methyl, or Rla, R2a, R3a and R4a are H and R1 and R2 or R2 and R3 are taken together to form a C4-C5 cycloalkyl ring, or, when n is 0, R1, Rla, R2a, R3\ R4 and R4a are H and R2 and R3 form a cyclopentyl ring, or, when n is 1, R1, Rla, R2a, R3a, R4 and R4a are H and R2 and R3 are both methyl or R1, Rla, R2a, 25 R3a, R4 and R4a are H and R2 and R3 form a cyclobutyl ring, or, when n is 2, R1, Rla,R2, R20 * * 23 * 25, R3, R3a, R4 and R4a are H, or, n is 0, R1, Rla, R2a, R3a, R4 and R4a are H and R2and R form a cyclopentyl ring.

Useful acyclic alpha-2-delta ligands of the présent invention may be depicted

wherein: 5 n is 0 or 1, R1 is hydrogen or (Q-C^alkyl; R2 is hydrogen or (Ci-C6)alkyl; R3 is hydrogen or (Ci-C6)alkyl; R4 is hydrogen or (Ci-Cô)alkyl; R5 is hydrogen or (Ci-Cg)alkyl and R2 is hydrogen or (C]-C6)alkyl, or a pharmaceutically acceptable sait or ' solvaté thereof. LO According to formula (H), suitably R1 is Ci-Cô alkyl, R2 is methyl, R3 - R6 are hydrogen and n is 0 or 1. More suitably R1 is methyl, ethyl, n-propyl or n-butyl, R2 ismethyl, R3 - R6 are hydrogen and n is 0 or 1. When R2 is methyl, R3 - R6 arehydrogen and n is 0, R1 is suitably ethyl, n-propyl or n-butyl. When R2 is methyl, R3- R6 are hydrogen and n is 1, R1 is suitably methyl or n-propyl. Compounds of L5 formula (Π) are suitably in the 3S,5R configuration.

Examples of PDEV inhibitors for use with the invention are: the pyrazolo [4,3- ? d]pyrimidin-7-ones disclosed in EP-A-0463756; the pyrazolo [4,3-d]pyrimidin-7-onesdisclosed in EP-A-0526004; the pyrazolo [4,3-d]pyrimidin-7-ones disclosed in 20 published international patent application WO 93/06104; the isomeric pyrazolo [3,4-d]pyrimidin-4-ones disclosed in published international patent application WO93/07149; the quinazolin-4-ones disclosed in published international patentapplication WO 93/12095; the pyrido [3,2-d]pyrimidin-4-ones disclosed in publishedinternational patent application WO 94/05661; the purin-6-ones disclosed in 25 published international patent application WO 94/00453; the pyrazolo [4,3-d]pyrimidin-7-ones disclosed in published international patent application WO98/49166; the pyrazolo [4,3-d]pyrimidin-7-ones disclosed in published internationalpatent application WO 99/54333; the pyrazolo [4,3-d]pyrimidin-4-ones disclosed inEP-A-0995751; the pyrazolo [4,3-d]pyrimidin-7-ones disclosed in published 012899 8 international patent application WO 00/24745; the pyrazolo [4,3-d]pyrimidin-4-onesdisclosed in EP-A-0995750; the hexahydropyrazino [2’,l’:6,l]pyrido [3,4-h]indole- 1,4-diones disclosed in published international application WO95/19978; theimidazo[5,l-/][l,2,4]triazin-ones disclosed in EP-A-1092719 and in published 5 international application WO 99/24433 and the bicyclic compounds disclosed inpublished international application WO 93/07124, ail of which are incorporatedherein by reference.

Further examples of suitable PDEV inhibitors for use herein include: the 10 pyrazolo [4,3-d]pyrimidin-7-ones disclosed in published international application WO01/27112; the pyrazolo [4,3-d]pyrimidin-7-ones disclosed in published internationalapplication WO 01/27113; the compounds disclosed in EP-A-1092718 and thecompounds disclosed in EP-A-1092719; the tricyclic compounds disclosed in EP-A-1241170; the alkyl sulphone compounds disclosed in published international 15 application WO 02/074774; the compounds disclosed in published internationalapplication WO 02/072586; the compounds disclosed in published internationalapplication WO 02/079203 and the compounds disclosed in WO 02/074312, ail ofwhich are incorporated herein by reference. 20 Yet further examples of suitable PDEV inhibitors for use herein include: the carboline dérivatives described in W003000691, WO02098875, W002064591,W002064590 and W00108688, the pyrazino [l’,2’:l,6] pyrido [3,4-B] indole 1,4-dione dérivatives described in WO02098877, the tetracyclic compounds described inWO02098428, the compounds described in W002088123 and W00200656, the 25 condensed pyrazindione dérivatives described in WO0238563 and W002000657, the( indole dérivatives described in WO0236593, the condensed pyrindole dérivativesdescribed in WO0228865 and WO0228859, the hexahydropyrazino[l’,2’:l,6]-pyrido[3,4-B] indole-1,4-dione dérivatives described in WO0228858 and WO0194345, thefused heterocyclic dérivatives described in W00210166, the cyclic GMP spécifie 30 phosphodiesterase inhibitors described in W00200658, the tetracyclicdiketopiperazine compounds described in WOO194347 and the compounds describedin use application WO0219213, ail of which are incorporated herein by reference. 012899

Still other PDEV inhibitors inhibitors useful in conjunction with the présentinvention include: 4-bromo-5-(pyridylmethylamino)-6-[3-(4-chlorophenyl)-propoxy3-3(2H)pyridazinone; l-[4-[(l,3-benzodioxol-5- ylmethyl)amiono]-6-chloro-2- 5 quinozolinyl]-4-piperidine-carboxylic acid, monosodium sait; (+)-cis-5,6a,7,9,9,9a-hexahydro-2-[4-(trifluoromethyl)-phenylmethyl-5-methyl-cyclopent-4,5]imidazo[2,l-b]purin-4(3H)one; furazlocillin; cis-2-hexyl-5-methyl-3,4,5,6a,7,8,9,9a- octahydrocyclopent[4,5]-imidazo[2,l-b]purin-4-one; 3-acetyl-l-(2-chlorobenzyl)-2-propylindole-6- carboxylate; 3-acetyl-l-(2-chlorobenzyl)-2-propylindole-6- 10 carboxylate; 4-bromo-5-(3-pyridylmethylamino)-6-(3-(4-chlorophenyl) propoxy)-3-(2H)pyridazinone; l-methyl-5(5-morpholinoacetyl-2-n-propoxyphenyl)-3-n-propyl-1,6-dihydro- 7H-pyrazolo(4,3-d)pyrimidin-7-one; l-[4-[(l,3-benzodioxol-5-ylmethyl)amino]-6-chIoro-2- quinazolinyl]-4-piperidinecarboxylic acid, monosodiumsait; Pharmaprojects No. 4516 (Glaxo Wellcome); Pharmaprojects No. 5051 (Bayer); 15 Pharmaprojects No. 5064 (Kyowa Hakko; see WO 96/26940); Pharmaprojects No.5069 (Schering Plough); GF-196960 (Glaxo Wellcome); E-8010 and E-4010 (Eisai);Bay-38-3045 & 38-9456 (Bayer); FR229934 and FR226807 (Fujisawa); and Sch-51866. 20 Preferred PDEV inhibitors for the use according to the présent invention ,<"'j include: (i) 5-[2-ethoxy-5-(4-methyl-l-piperazinylsulphonyl)phenyl]-l-methyl-3-n-propyl-l,6-dihydro-7H-pyrazolo[4,3-dlpyrimidin-7-one (sildenafil) also known as 1-[[3-(6,7-dihydro-l-methyl-7-oxo-3-propyl-lH-pyrazolo[4,3-d]pyrimidin-5-yl)-4- 25 ethoxyphenyl]sulphonyl]-4-methylpiperazine (see EP-A-0463756); (ii) 5-(2-ethoxy-5-morpholinoacetylphenyl)-l-methyl-3-n-propyl-l,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (see EP-A-0526004); (iii) 3-ethyl-5-[5-(4-ethylpiperazin-l-ylsulphonyl)-2-n-propoxyphenyl]-2- (pyridin-2-yl)methyî-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (see 30 WO98/49166); 012899 10 (iv) 3-ethyl-5-[5-(4-ethylpiperazin- l-ylsulphonyl)-2-(2-methoxyethoxy)pyridin-3-yl]-2-(pyridin-2-yl)methyl-2,6-dîhydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (see WO99/54333); (v) (+)-3-ethyl-5-[5-(4-ethylpiperazin-l-ylsulphonyl)-2-(2-methoxy-l(R)-5 methylethoxy)pyridin-3-yl]-2-methyl-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7- one, also known as 3-ethyl-5-{5-[4-ethylpiperazin-l-ylsulphonyl]-2-([(lR)-2-methoxy- 1-methylethyl] oxy)pyridin-3 -yl} -2-methyl-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (see WO99/54333); (vi) 5-[2-ethoxy-5-(4-ethylpiperazin-l-ylsulphonyl)pyridin-3-yl]-3-ethyl-2-[2-10 methoxyethyl]-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one, also known as l-{6- ethoxy-5-[3-ethyl-6,7-dihydro-2-(2-methoxyethyl)-7-oxo-2H-pyrazolo[4,3-d]pyrimidin-5-yl]-3-pyridylsulphonyl}-4-ethylpiperazine (see WO 01/27113,Example 8); (vii) 5-[2-iso-Butoxy-5-(4-ethylpiperazin-l-ylsulphonyl)pyridin-3-yl]-3-ethyl-15 2-(l-methylpiperidin-4-yl)-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (see WO 01/27113, Example 15); (viii) 5-[2-Ethoxy-5-(4-ethylpiperazin-l-ylsulphonyl)pyridin-3-yl]-3-ethyl-2-phenyl-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (see WO 01/27113, Example66); 20 (ix) 5-(5-Acetyl-2-propoxy-3-pyridinyl)-3-ethyl-2-(l-isopropyl-3-azetidinyl)- 2,6-dihydro-7ZZ-pyrazolo[4,3-ii]pyrimidin-7-one (seeWO 01/27112, Example 124); (x) 5-(5-Acetyl-2-butoxy-3-pyridinyl)-3-ethyl-2-(l-ethyI-3-azetidinyl)-2,6-dihydro-7//-pyrazolo[4,3-<Z]pyriinidin-7-one (see WO 01/27112, Example 132); (xi) (6R,12aR)-2,3,6,7,12,12a-hexahydro-2-methyl-6-(3,4-25 methylenedioxyphenyl) -pyrazino[2',r:6,l]pyrido[3,4-b]indole-l,4-dione (tadalafil, IC-351, Cialis®), i.e. the compound of examples 78 and 95 of published internationalapplication WO95/19978, as well as the compound of examples 1,3,7 and 8; (xii) 2-[2-ethoxy-5-(4-ethyl-piperazin-l-yl-l-sulphonyl)-phenyl]-5-methyl-7-propyl-3H-imidazo[5,l-f][l,2,4]triazin-4-one (vardenafil) also known as l-[[3-(3,4- 30 dihydro-5-methyl-4-oxo-7-propylimidazo[5, l-f]-as-triazin-2-yl)-4- ethoxyphenyl]sulphonyl]-4-ethylpiperazine, i.e. the compound of examples 20, 19,337 and 336 of published international application WO99/24433; 012899 11 (xiii) the pyrazolo [4,3-d]pyrimidin-4-ones disclosed in WOOO/27848, inparticular N-[[3-(4,7-dihydro-l-methyl-7-oxo-3-propyl-lH-pyrazolo[4,3-d]- pyrimidin-5-yl)-4-propxyphenyl]sulfonyl]-l-methyl2-pyrrolidinepropanamide [DA-8159 (Example 68 of WOOO/27848)]; 5 (xiv) the compound of example 11 of published international application WO93/07124; (xv) 4-(4-chlorobenzyl)amino-6,7,8-trimethoxyquinazoline; and (xvi) 7,8-dihydro-8-oxo-6-[2-propoxyphenyl]-lH-imidazo[4,5-g]quinazoline; (xvii) l-[3-[l-[(4-fluorophenyl)methyl]-7,8-dihydro-8-oxo-lH-imidazo[4,5- 10 g]quinazoiin-6-yl]-4-propoxyphenyl]carboxamide; (xviii) 5-(5-acetyl-2-butoxy-3-pyridinyl)-3-ethyl-2-(l-ethyb3-azetidinyl)-2,6-' drhydro-7fl-pyrazolo[4,3--d]pyrimidin-7-one; and (xix) l-{6-ethoxy-5-[3-ethyl-6,7-dihydro-2-(2-methoxyethyl)-7-oxo-2H-pyrazolo [4,3- d]pyrimidin-5-yl] -3-pyridylsulfonyl} -4-ethylpiperazine; and 15 pharmaceutically acceptable salts and solvatés thereof.

The suitability of any particular PDEV inhibitor can be readily determined byévaluation of its potency and selectivity using literature methods followed byévaluation of its toxicity, absorption, metabolism, pharmacokinetics, etc in 20 accordance with standard pharmaceutical practice.

Preferably, the PDEV inhibitors hâve an IC50 at less than 100 nanomolar, morepreferably, at less than 50 nanomolar, more preferably still at less than 10 nanomolar. 25 IC50 values for the PDEV inhibitors may be determined using the PDE5 assay described hereinafter.

Preferably the PDEV inhibitors used in the pharmaceutical combinationsaccording to the présent invention are sélective for the PDEV enzyme. Preferably 30 they hâve a selectivity of PDEV over PDE3 of greater than 100 more preferablygreater than 300. More preferably the PDEV inhibitor has a selectivity over bothPDE3 and PDE4 of greater than 100, more preferably greater than 300. Selectivity 012899 12 ratios may readily be detennined by the skilled person. IC50 values for the PDE3 andPDE4 enzyme may be determined using established literature methodology, see S ABallard et al, Journal of Urology, 1998, vol. 159, pages 2164-2171 and as detailedherein after.

Useful PDEV inhibitors of the présent invention may be depicted by thefollowing formula (ΙΠ):

10 wherein: „ A is CH or N; R1 is H, Ci to C6 alkyl, C3 to C<, alkenyl, C3 to C6 cycloalkyl, C3 to Cô cycloalkenyl, or Ci-C3 perfluoroalkyl, wherein said alkyl group may be branched or straight Chain and wherein said alkyl, alkenyl, cycloalkyl or perfluoroalkyl group is 15 optionally substituted by; one or more substituents selected from: hydroxy; Ci to C4 alkoxy; C3 to C6 cycloalkyl; Ci-C3 perfluoroalkyl; phenyl substituted with one or more substitutents selected from Ci to C3 alkyl, Ci to C4 alkoxy, C] to C4 haloalkyl or

Ci to C4 haloalkoxy wherein said haloalkyl and haloalkoxy groups contain one or more halo atoms, halo, CN, NO2, NHR10 11, NHSO2R12 * * 15 * * * * 20 * * * * 25, SO2R12, SO2NHRU, COR11, 20 CO2R11 wherein R11 is H, Ci to C4 alkyl, C2 to C4 alkenyl, Ci to C4 alkanoyl, Ci to C4 haloalkyl or Ci to C4 haloalkoxy and wherein R12 is Ci to C4 alkyl, C2 to C4 alkenyl,

Ci to C4 alkanoyl, Ci to C4 haloalkyl or Ci to C4 haloalkoxy; NR7R8, CONR7R8 or NR7CORH wherein R7 and R8 are each independently selected from H, Ci to C4 alkyl, C3 to C4 alkenyl, Ci to C4 alkoxy, CO2R9, SO2R9 wherein said alkyl, alkenyl or 25 alkoxy groups are optionally substituted by NR5R6, Ci to C4 haloalkyl or Ci to C4 haloalkoxy and wherein R9 is H, hydroxy C2 to C3 alkyl, Ci to C4 alkanoyl or Ci to C4alkyl which is optionally substituted with phenyl wherein said phenyl group is 13 optionally substituted by one or more substituents selected from Ci to C4 alkyl optionally substituted by Ci to C4 haloalkyl or Ci to C4 haloalkoxy, Ci to C4 alkoxy, halo, CN, NO2, NHR11, NHSO2R12, SO2R12, SO2NHR11, COR11 or CO2Rn; Het1;

Het2 or Het3; or R1 is Het4 or phenyl wherein said phenyl group is optionally 5 substituted by one or more substituents selected from Ci to C4 alkyl, C2 to C4 alkenyl,C, to C4 alkoxy, halo, CN, CF3, OCF3, NO2, NHR11, NHSO2R12, SO2R12, SO2NHR11,COR11, CO2Rn; R2 is H, Ci to Cô alkyl, C3 to C6 alkenyl or (CH2)n(C3 to C6 cycloalkyl)wherein n is 0,1 or 2 and wherein said alkyl or alkyenyl group is optionally 10 substituted with one or more fluoro substituents; R13isOR3 orNRsR6; R3 is Ci to Ce alkyl, C3-C6 alkenyl, C3-C6 alkynyl, C3-C7 cycloalkyl, Ci-Cëperfluoroalkyl or (C3-Cë cycloalkyl)Ci-Ce alkyl optionally substituted with one or twosubstituents selected from C3 to C5 cycloalkyl, hydroxy, Ci to C4 alkoxy, C3-Cë 15 alkenyl, C3-Cë alkynyl, benzyloxy, NRSR6, phenyl, Het1, Het2, Het3 or Het4 whereinthe Ci to C6 alkyl and Ci to C4 alkoxy groups may optionally be terminated by ahaloalkyl group such as CF3; C3 to C5 cycloalkyl; Het1, Het2, Het3 or Het4; R4is C]-C4 alkyl optionally substituted with OH, NfCR6, CN, CONR5R6 orCO2R7; C2-C4 alkenyl optionally substituted with CN, CONR5R6 or CO2R7; C2-C4 20 alkanoyl optionally substituted with NRSR6; hydroxy C2-C4 alkyl optionally - ( substituted with NRSR6; (C2-C3 alkoxy)Ci-C2 alkyl optionally substituted with OH or NR5R6; CONR5R6; CO2R7; halo; NR5R6; NHSO2NR5R6; NHSO2R8; or phenyl orheterocyclyl either of which is optionally substituted with methyl; or R4 is apyrrolidinylsulphonyl, piperidinosulphonyl, morpholinosulphonyl, orpiperazin-1- 25 ylsulphonyl group having a substituent, R10 at the 4-position of the piperazinyl groupwherein said piperazinyl group is optionally substituted with one or two Ci to C4alkyl, Ci to C3 alkoxy, NR7R8 or CON R7R8 groups and is optionally in the form of its4-N-oxide; R5 and R6 are each independently selected from H and Ci to C4 alkyl 30 optionally substituted with C3 to C5 cycloalkyl or Ci to C4 alkoxy, or, together withthe nitrogen atom to which they are attached, form an azetidinyl, pynolidinyl, 012899 14 piperidinyl, morpholinyl, 4-(NR9)- piperazinyl or imidazolyl group wherein saidgroup is optionally substituted with methyl or hydroxy; R10 is H; Ci to Cô alkyl, (C1-C3 alkoxy) C2-Cô alkyl, hydroxy C2-Cô alkyl,(R7R8N)C2-C6 alkyl, (R7R8NCO)C1-C6 alkyl, CONR7R8, CSNR7R8 or C(NH)NR7R8 5 optionally substituted with one or two substituents selected front hydroxy, NR5R6,CONRSR6, phenyl optionally substituted with Ci to C4 alkyl or Ci to C4 alkoxy; C2 toCe alkenyl or Het4;

Het1 is an N-linked 4-, 5- or 6-membered nitrogen-containing heterocyclicgroup optionally containing one or more further heteroatoms selected from S, N or O;

10 Het2 is a C-linked 5-membered heterocyclic group containing an O, S or N heteroatom optionally containing one or more heteroatoms selected from O or S;

Het3 is a C-linked 6-membered heterocyclic group containing an O or Sheteroatom optionally containing one or more heteroatoms selected from O, S or N orHet3 is a C-linked 6-membered heterocyclic group containing three N heteroatoms; 15 Het4 is a C-linked 4-, 5- or 6-membered heterocyclic group containing one,. two or three heteroatoms selected from S, O or N; and wherein any of saidheterocyclic groups Het1, Het2, Het3 or Het4 may be saturated, partially unsaturated oraromatic and wherein any of said heterocyclic groups may be optionally substitutedwith one or more substituents selected from Ci to C4 alkyl, C2 to C4 alkenyl, Ci to C4 20 alkoxy, halo, CO2R11, COR11, SO2R12 or NHR11 and/or wherein any of saidheterocyclic groups is benzo-fused; or wherein when R13 represents OR3 or R3NR5; R1 represents Het, alkylHet,aryl or alkylaryl, which latter five groups are ail optionally substituted and/orterminated with one or more substituents selected from halo, cyano, nitro, lower alkyl, 25 halo(loweralkyl), OR6, OC(O)R7, C(O)R8, C(O)OR9, C(O)NR10Rn, NR12R13 andSO2NR14R15; R2 represents H, halo, cyano, nitro, OR6, OC(O)R7, C(O)R8, C(O)OR9,C(O)NR10Rn, NR12R13, SO2NR14R15, lower alkyl, Het, alkylHet, aryl or alkylaryl,which latter five groups are ail optionally substituted and/or terminated with one ormore substituents selected from halo, cyano, nitro, lower alkyl, halo(loweralkyl), OR6, 30 OC(O)R7, C(O)R8, C(O)OR9, C(O)NR10R11, NR12R13 and SO2NR14R15; R3 representsH, lower alkyl, alkylHet or alkylaryl, which latter three groups are ail optionallysubstituted and/or terminated with one or more substituents selected from halo, cyano, 012899

15 20 25 30 15 nitro, lower alkyl, halo(loweralkyl), OR6, OC(O)R7, C(O)R8, C(O)OR9, C(O)NR10Rn,NR12R13 and SO2NR14R15; R4 represents H, halo, cyano, nitro, halo(loweralkyl), OR6,OC(O)R7, C(O)R8, C(O)OR9, C(O)NRlcfen, NRI2R13, NR16Y(O)R17, SOR18,SO2R19R20, C(O)AZ, lower alkyl, lower alkenyl, lower alkynyl, Het, alkylHet, aryl,alkylaryl, which latter seven groups are all optionally substituted and/or terminatedwith one or more substituents selected from halo, cyano, nitro, lower alkyl,halo(loweralkyl), OR6, OC(O)R7, C(O)R8, C(O)OR9, C(O)NR10R11, NR12R13 andSO2NR14R15; Y represents C or S(O), wherein one of R16 and R17 is not présent whenY is S(O); A represents lower alkylene; Z represents OR6, halo, Het or aryl, whichlatter two groups are both optionally substituted with one or more substituentsselected from halo, cyano, nitro, lower alkyl, halo(loweralkyl), OR6, 0C(O)R7,O(O)R8, C(O)OR9, C(O)NR10Rn, NR12R13 and SO2NR14R15; R5, R6, R7, R8, R9, R18,R19 and R20 independently represent H or lower alkyl; R10 and R11 independentlyrepresent H or lower alkyl, which latter group is optionally substituted and/orterminated with one or more substituents selected from halo, cyano, nitro, lower alkyl,halo(loweralkyl), OR6, OC(O)R7, C(O)R8, C(O)OR9, C(O)NR10Rn, NR12R13 andSO2NR14R15 or Het or aryl optionally substituted with one or more of said lattereleven groups or one of R10 and R11 may be lower alkoxy, amino or Het, which lattertwo groups are both optionally substituted with lower alkyl; R12 and R13independently represent H or lower alkyl or one of R12 or R13 may be C(O)-loweralkyl or C(O)Het in which Het is optionally substituted with lower alkyl; R14 and R15independently represent H or lower alkyl or R14 and R15, together with the nitrogenatom to which they are bound, form a heterocyclic ring; R16 and R17 independentlyrepresent H or lower alkyl or one of R16 and R17 may be Het or aryl, which latter twogroups are both optionally substituted with lower alkyl; Het represents an optionallysubstituted four to twelve membered heterocyclic group, which may be aromatic ornon-aromatic, which may contain one or more double bonds, which may be mono- orbi-cyclic and which contains one or more heteroatoms selected from N, S and O;or a pharmaceutically acceptable sait or solvaté of any thereof.

In formula (DI), the PDEV inhibitor may contain halo groups. Here, “halo”means fluoro, chloro, bromo or iodo. 16 012899

In formula (Ht), the PDE5 inhibitor may contain one or more of alkyl, alkoxy,alkenyl, alkylene and alkenylene groups - which may be unbranched- or branched-chain. 5

In formula (ΠΙ), a preferred group of compounds for use according to theprésent invention are those wherein: R1 is H, methyl or ethyl; R2 is H, C1-C3 alkyloptionally substituted by OH, or methoxy; R is C2-C3 alkyl or allyl; R is asulphonylpiperidino or 4-N-(R10)-sulphonylpiperazin-l-yl group; R5 is H, NR7R8, or 10 CONR7R8; R10 is H, C1-C3 alkyl, hydroxy C2-C6 alkyl, CONR7R8, CSNR7R8 orC(NH)NR7R8; R7 and R8 are each independently H or methyl.

In formula (ΠΙ), another preferred group of compounds for use according tothe présent invention are those wherein: R1 is Ci to C2 alkyl optionally substituted 15 with Het; 2-(morpholin-4-yl)ethyl or benzyl; R2 is C2 to C4 alkyl; R13 is OR3 or NR5R6; R3 is Ci to C4 alkyl optionally substituted with one or two substituentsselected from cyclopropyl, cyclobutyl, OH, methoxy, ethoxy, benzyloxy, NR5R6,phenyl, furan-3-yl, pyridin-2-yl and pyridin-3-yl; cyclobutyl; l-methylpiperidin-4-yl;tetrahydrofuran-3-yl or tetrahydropyran-4-yl; R5 and R6 are each independently 20 selected from H and Ci to C2 alkyl optionally substituted with cyclopropyl ormethoxy, or, together with the nitrogen atom to which they are attached, form aazetidinyl, pyrrolidinyl or morpholinyl group; R7 and R8, together with the nitrogenatom to which they are attached, form a4-R10-piperazinyl group optionally substitutedwith one or two methyl groups and optionally in the form of its 4-N-oxide; R10 is H, 25 Ci to C3 alkyl optionally substituted with one or two substituents selected from OH,NR5R6, CONR5R6, phenyl optionally substituted with methoxy, benzodioxol-5-yl andbenzodioxan-2-yl; allyl; pyridin-2-yl; pyridin-4-yl or pyrimidin-2-yl; and Het isselected from pyridin-2-yl; l-oxidopyridin-2-yl; 6-methylpyridin-2-yl; 6-methoxypyridin-2-yl; pyridazin-3-yl; pyrimidin-2-yl and l-methylimidazol-2-yl. Of 30 this group more preferred are those compounds wherein R1 is Ci to C2 alkyl optionally substituted with Het; 2-(morpholin-4-yl)ethyl or benzyl; R2 is C2 to C4alkyl; R13 is OR3; R3 is Ci to C4 alkyl optionally monosubstituted with cyclopropyl, 012899 17 cyclobutyl, OH, methoxy, ethoxy, phenyl, furan-3-yl or pyridin-2-yl;1 cyclobutyl;tetrahydrofuran-3-yl or tetrahydropyran-4-yl; R7 and R8, togetfier with the nitrogenatom to which they are attached, form a 4-R10-piperazinyl group optionally in theform of its 4-N-oxide; R10 is Ci to C3 alkyl optionally monosubstituted with OH; and 5 Het is selected from pyridin-2-yl; l-oxidopyridin-2-yl; 6-methylpyridin-2-yl; 6-methoxypyridin-2-yl; pyridazin-3-yl; pyrimidin-2-yl and l-methylixnidazol-2-yl.

15 20 25

In formula (TU), one other further preferred group of compounds for useaccording to the présent invention are those wherein: R1 is Ci to C6 alkyl or C3 to C<5alkenyl wherein said alkyl or alkenyl groups may be branched chain or straight chainor R1 is C3 to Cg cycloalkyl or C4 to C6 cycloalkenyl and wherein when R1 is Ci to C3alkyl said alkyl group is substituted by; and wherein when R1 is C4 to C6 alkyl, C3 toCô alkenyl, C3 to Cg cycloalkyl or C4 to Ce cycloalkenyl said alkyl, alkenyl, cycloalkylor cycloalkenyl group is optionally substituted by; one or more substituents selectedfrom: hydroxy; Ci to C4 alkoxy; C3 to C4 cycloalkyl; phenyl substituted with one ormore substitutents selected from Ci to C3 alkyl, Ci to C4 alkoxy, Ci to C4 haloalkyl orCi to C4 haloalkoxy, halo, CN, NO2, NHR11, NHCOR12, NHSO2R12, SO2R12,SO2NHR”, COR11, CO2R11 wherein said haloalkyl and haloalkoxy groups containone or more halo atoms; NR7R8, CONR7R8 or NR7CORn; a Het1 group which is anN-Iinked 4-membered N-containing heterocyclic group; a Het2 group which is a C-linked 5-membered heterocyclic group containing an O, S or N heteroatom optionallycontaining one or more heteroatoms selected from N, O or S; a Het3 group which is aC-linked 6-membered heterocyclic group containing an O or S heteroatom optionallycontaining one or more heteroatoms selected from O, S or N or a Het3 group which isa C-linked 6-membered heterocyclic group containing three N heteroatoms; whereinR7, R8, R11 and R12 are as previously defined herein or R1 is a Het4 group which is aC-linked 4- or 5-membered heterocyclic group containing one heteroatom selectedfrom S, O or N; a Het4 group which is a C-linked 6-membered heterocyclic groupcontaining one, two or three heteroatoms selected from S or O; a Het4 group which isa C-linked 6-membered heterocyclic group containing three nitrogen heteroatoms; aHet4 group which is a C-linked 6-membered heterocyclic group containing one or twonitrogen heteroatoms which is substituted by one or more substitutents selected from 30 18

Ci to C4 alkyl, Ci to C4 alkoxy, CO2Rn, SO2R12, COR11, NHR11 or NHCOR12 andoptionally including a further heteroatom selected from S, O or N wherein any of saidheterocyclic groups Het1, Het2, Het3 or Het4 is saturated, partially unsaturated oraromatic as appropriate and wherein any of said heterocyclic groups is optionally 5 substituted with one or more substituents selected from Ci to C4 alkyl, C3 to C4alkenyl, Ci to C4 alkoxy, halo, CO2R11, SO2R12, COR11 or NHR11 wherein R11 is asdefined hereinbefore and/or wherein any of said heterocyclic groups is benzo-fused;or R1 is phenyl substituted by one or more substituents selected from CF3, OCF3,SO2R12 or CO2R12 wherein R12 is Ci to C4 alkyl which is optionally substituted by 10 phenyl, Ci to C4 haloalkyl or Ci to C4 haloalkoxy wherein said haloalkyl and haloalkoxy groups contain one or more halo atoms; R2 is Ci to Cé alkyl; R13 is OR3; R3 is Ci to Cé alkyl optionally substituted with one or two substituents selected fromC3 to C5 cycloalkyl, hydroxy, Ci to C4 alkoxy, benzyloxy, NR5R6, phenyl, furanyl,tetrahydrofuranyl or pyridinyl wherein said Ci to Ce alkyl and Ci to C4 alkoxy groups 15 may optionally be terminated by a haloalkyl group such as CF3; or R3 is C3 to Cô cycloalkyl, l-(Ci to C4 alkyl)piperidinyl, tetrahydrofuranyl or tetrahydropyranyl; R4 isa piperazin-1-ylsulphonyl group having a substituent R10 at the 4-position of thepiperazinyl group wherein said piperazinyl group is optionally substituted with one ortwo Ci to C4 alkyl groups and is optionally in the form of its 4-N-oxide; R5 and R6 are 20 each independently selected from H and Ci to C4 alkyl optionally substituted with C3to C5 cycloalkyl or Ci to C4 alkoxy, or, together with the nitrogen atom to which theyare attached, form an azetidinyl, pyrrolidinyl, piperidinyl or morpholinyl group; andR10is H; Ci to C4 alkyl optionally substituted with one or two substituents selectedfrom hydroxy, NR5R6, CONR5R6, phenyl optionally substituted with Ci to C4 alkyl or 25 Ci to C4 alkoxy; C3 to alkenyl; Het4; with the proviso that when R1 is Ci to C3alkyl substituted by phenyl then said phenyl group is not substituted by Ci to C4alkoxy; CN; halo; CF3; OCF3; or Ci to C4 alkyl. More preferred of this group ofcompounds are those wherein R1 is Ci to C6 alkyl wherein said alkyl may be branchedor straight chain or R1 is C3 to C6 cycloalkyl and wherein when R1 is Ci to C3 alkyl 30 said alkyl group is substituted by; and wherein when R1 is C4 to C6 alkyl or C3 to C6cycloalkyl said alkyl or cycloalkyl group is optionally substituted by; one or moresubstituents selected from·. hydroxy; Ci to C2 alkoxy; C3 to C5 cycloalkyl; NR7R8, 012899 19

NR7CORn or COR11 wherein R7 and R8 are each independently selected from H, Cjto C4 alkyl or CO2R9 wherein R9 and R11 are as previously defined herein; a Het1group which is an N-linked 4-membered N-containing heterocyclic group; a Het3group which is a C-linked 6-membered heterocyclic group containing an O or S 5 heteroatom optionally containing one or more heteroatoms selected from O, S or N ora Het3 group which is a C-linked 6-membered heterocyclic group containing three Nheteroatoms; or R1 is a Het4 group which is a C-linked 4-membered heterocyclicgroup containing one heteroatom selected from S, O or N or R1 is a Het4 group whichis a C-linked 6-membered heterocyclic group containing one, two or three jlo heteroatoms selected from S or O wherein any of said heterocyclic groupe Het1, Het2,Het3 or Het4 is saturated, partially unsaturated or aromatic and is optionallysubstituted with one or more substituents selected from Ci to C4 alkyl, Ci to C4alkoxy, -CO2Rn, -SO2R12, -COR11 orNHR11 wherein R11 and R12 are as definedhereinbefore and/or wherein any of said heterocyclic groupe is benzo-fused; or R1 is 15 phenyl substituted by one or more substituents selected from : CF3, -OCF3, -SO2R12, -COR11, -CO2R11 wherein R11 and R12 are as defined hereinbefore; R2 is Ci to C6alkyl; R13 is OR3; R3 is methyl, ethyl, n-propyl, i-propyl, n-butyl, sec-butyl, i-butyl ort-butyl alkyl optionally substituted with one or two substituents selected from *cyclopropyl, cyclobutyl, hydroxy, methoxy, ethoxy, benzyloxy, phenyl, benzyl, furan- 20 3-yl, tetrahydrofuran-2-ylmethyl, tetrahydrofuran-3-ylmethyl, pyridin-2-yl, pyridin-3- * , ylorNR5R6 wherein R5 and R6 are each independently selected from H and Ci to C2 alkyl; R4 is a piperazin-1-ylsulphonyl group having a substituent, R10 at the 4-positionof the piperazinyl group wherein said piperazinyl group is optionally substituted withone or two Ci to C4 alkyl groups and is optionally in the form of its 4-N-oxide; and 25 R10 is H, Ci to C3 alkyl optionally substituted with one or two substituents selected from hydroxy, NR5R6, CONR5R6 wherein R5 and R6 are each independently selectedfrom H, Ci to C4 alkyl and C3 alkenyî.

In formula (IH), a further group of preferred compounds for use according to 30 the présent invention are those wherein: R1 représente H, lower alkyl, Het, alkylHet,or alkylaryl (which latter four groups are ail optionally substituted and/or terminatedwith one or more substituents selected from cyano, lower alkyl, OR6, C(0)OR9 or 012899 20 NR12R13); R2 represents H, halo, lower alkyl, Het or aryl (which latter three groupsare ail optionally substituted and/or terminated with one or more substituents asdefîned hereinbefore, and preferably with NR12R13 or SO2NR14R15); R3 represents Ci-C4 alkyl or C3-C4 cycloalkyl which are optionally substituted and/or terminated with 5 one or more substituents selected from halo, cyano, nitro, lower alkyl,halo(loweralkyl), OR6, OC(O)R7, C(O)R8, C(O)OR9, C(O)NRiaRn, NRI2R13 andSO2NR14R15); R4 represents halo, cyano, nitro, C(O)R8, C(O)OR9, C(O)NR10Ru,NR12R13, N[Y(O)R17]2, NR16Y(O)R17, SOR18, SO2R19, C(O)AZ, lower alkyl, lower alkynyl, Het or aryl, which latter three groups are ail optionally substituted and/or 10 terminated with one or more substituents as defîned hereinbefore; and wherein Y, A,Z, R10, R11, R12, R13, R14, R15, R16, R17, R5, R6, R7, R8, R9, R18, R19 and Het are as herein before defîned. More preferred in this further group are compounds inwhich R1 represents optionally substituted lower alkyl, more preferably lower alkyl,lower alkoxy-teiminated lower alkyl, NR12R13-terminated lower alkyl, or N- 15 morpholino-terminated lower alkyl. Altematively, R1 may represent a 4-piperidinylor a 3-azetidinyl group, optionally substituted at the nitrogen atom of the piperidinylgroup with lower alkyl or C(O)OR9. In such more preferred compounds in thisfurther group R2 represents C(O)NR10Rn, NR12R13, lower alkyl optionally interruptedby one or more of O, S or N, optionally substituted at N by lower alkyl or acyl, or 20 optionally substituted aryl or Het. More preferably, when R2 is interrupted loweralkyl, the interrupting atoms are one or more of O and lower alkylated-N and when R2is aryl, it is optionally substituted phenyl or pyridyl. Particularly preferredcompounds of this further group are those in which R2 represents C(O)NR10Rn,NRi2R13, Cm alkyl optionally interrupted by O or N, optionally substituted at N by 25 lower alkyl, optionally substituted phenyl, or optionally substituted pyridin-2-yl, ' pyridin-3-yl, pyrimidin-5-yl, pyrazin-2-yl, pyrazol-4-yl, oxadiazol-2-yl, furan-2-yl,furan-3-yl, tetrahydrofuran-2-yl and imidazo[l,2-a]pyridin-6-yl. In this morepreferred group of further compounds R3 may represent lower alkyl or cycloalkyl.Also, X is preferably O. Such further and more preferred compounds hâve R4 30 representing halo, lower alkyl, lower alkynyl, optionally substituted Het, optionallysubstituted aryl, C(O)R8, C(O)AZ, C(O)OR9, C(O)NR10R11, NRI2R13 or .17 NR16Y(O)R1Z. More preferred values for R* are C(O)R8 (e.g. acetyl), halo (e.g. iodo), 21 SO2R19 (wherein R19 represents lower alkyl) and C(O)NR10Rn (e.g. where Rlt} andR11 independently represent H and lower alkyl and/or one of R10 and R11 is loweralkoxy) or NHB, wherein B represents H, SO2CH3 or C(O)Het. Further preferred stillare compounds in which R4 represents iodo, lower alkyl, lower alkynyl (which latter 5 two groups are substituted and/or terminated by C(O)OR9 (wherein R9 represents H orCb6 alkyl)), N(H)Y(O)R17, N[Y(O)R17)2, optionally substituted Het or NR12R13(wherein R12 and R13 together represent C3.5 alkylene interrupted by O or N-S(O)2-(optionally substituted aryl)). ) 10 More preferred PDEV inhibitors for use with the invention, particularly with an alpha-2-delta ligand selected from gabapentin, pregabalin and (1α,3α,5α)(3-amino-methyl-bicyclo[3.2.0]hept-3-yl)-acetic acid, and pharmaceutically acceptablesalts or solvatés thereof, are selected from the group: 5-[2-ethoxy-5-(4-methyl-l-piperazinylsulphonyl)phenyl3-l-methyl-3-n-15 propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (sildenafîl); (6R, 12aR)-2,3,6,7,12,12a-hexahydro-2-methyl-6-(3,4-methylenediox yphenyl)-pyrazino[2',l':6,13pyrido[3,4-&]indole-l,4-dione (tadalafil, IC-351, Cialis®); 2-[2-ethoxy-5-(4-ethyl-piperazin- 1-yl- l-sulphonyl)-phenyl]-5-methyl-7-propyl-3774midazo[5,l-/][l,2,4]triazin-4-one (vardenafil); 20 5-[2-ethoxy-5-(4-ethylpiperazin-l-ylsulphonyl)pyridin-3-yl]-3-ethyl-2-[2- methoxyethyl]-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one; 5-(5-acetyl-2-butoxy-3-pyridinyl)-3-ethyl-2-(l-ethyl-3-azetidinyl)42,6-dihydro-7ff-pyrazolo[4,3-d]pyrimidin-7-oné; and l-{6-ethoxy-5-[3-ethyl-6,7-dihydro-2-(2-methoxyethyl)-7-oxo-2H-25 pyrazolo[4,3- d]pyrimidin-5-yl]-3-pyridylsulfonyI}-4-ethylpiperazine; and pharmaceutically acceptable salts and solvatés thereof A particularly preferred PDEV inhibitor, particularly with an alpha-2-deltaligand selected from gabapentin, pregabalin and (la.3a,5a)(3-amino-methyl- 30 bicyclo[3.2.0]hept-3-yl)-acetic acid, and pharmaceutically acceptable salts or solvatésthereof, is 5-[2-ethoxy-5-(4-methyl-l-piperazinylsulphonyl)phenyl]-l-methyl-3-n-propyl-1,6-dihydro-7f/-pyrazolo[4,3-J]pyrimidin-7-one (sildenafîl) and 012899 22 pharmaceutically acceptable salts or solvatés thereof. Sildenafil citrate is a preferredsait.

As an alternative or further aspect of the présent invention, there is provided a 5 combination, particularly a synergistic combination, comprising gabapentin and aPDEV inhibitor selected from sildenafil, l-{6-ethoxy-5-[3-ethyl-6,7-dihydro-2-(2-methoxyethyl)-7-oxo-2H-pyrazolo[4,3- d]pyrimidin-5-yl]-3-pyridylsulfonyl}-4-ethylpiperazine, 5-(5-acetyl-2-butoxy-3-pyridinyl)-3-ethyl-2-(l-ethyl-3-azetidinyl)-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one, vardenafil or tadalafil, or a 10 pharmaceutically acceptable sait or solvaté thereof. A particularly preferred combination comprises gabapentin and sildenafil or pharmaceutically acceptable saltsor solvatés thereof.

As an alternative or further aspect of the présent invention, there is provided a15 combination, particularly a synergistic combination, comprising pregabalin and a

PDEV inhibitor selected from sildenafil, l-{6-ethoxy-5-[3-ethyl-6,7-dihydro-2-(2-methoxyethyl)-7-oxo-2H-pyrazolo[4,3- d]pyrimidin-5-yl]-3-pyridylsulfonyl}-4-ethylpiperazine, 5-(5-acetyl-2-butoxy-3-pyridinyl)-3-ethyl-2-(l-ethyl-3-azetidinyl)-2,6-dihydro-72/-pyrazolo[4,3-<i|pyrimidin-7-one, vardenafil or tadalafil. A 20 particularly preferred combination comprises pregabalin and sildenafil.

As a yet further alternative or preferred aspect of the présent invention, there isprovided a combination, particularly a synergi stic combination, comprising[(lR,5R,6S)-6-(Aminomethyl)bicyclo[3.2.0]hept-6-yl]acetic acid or a 25 pharmaceutically acceptable sait or solvaté thereof, and a PDEV inhibitor. Suitably,there is provided a combination comprising [(lR,5R,6S)-6-(Aminomethyl)bicyclo[3.2.0]hept-6-yl]acetic acid or a pharmaceutically acceptablesait or solvaté thereof, and a PDEV inhibitor selected from sildenafil, l-{6-ethoxy-5-[3-ethyl-6,7-dihydro-2-(2-methoxyethyl)-7-oxo-2H-pyrazolo[4,3- d]pyrimidin-5-yI]- 30 3-pyridylsulfonyl}-4-ethylpiperazine, 5-(5-acetyl-2-butoxy-3-pyridinyl)-3-ethyl-2-(l-ethyI-3-azetidinyl)-2,6-dihydro-7if-pyrazolo[4,3-ti]pyrimidin-7-one, vardenafil or

23 tadalafil or a pharmaceutically acceptable sait or solvaté thereof, preferably sildenafil or a pharmaceutically acceptable sait or solvaté thereof.

Suitably, there is provided a combination comprising (la,3a,5a)(3-amino- 5 methyl-bicyclo[3.2.0]hept-3-yl)-acetic acid or a pharmaceutically acceptable sait orsolvaté thereof, and aPDEV inhibitor selected from sildenafil, l-{6-ethoxy-5-[3-ethyl-6,7-dihydro-2-(2-methoxyethyl)-7-oxo-2H-pyrazolo[4,3- d]pyrimidin-5-yl]-3-pyridylsulfonyl}-4-ethylpiperazine, 5-(5-acetyl-2-butoxy-3-pyridinyl)-3-ethyl-2-(l-ethyl-3-azetidinyl)-2,6-dihydro-7H-pyrazolo[4,3-i7]pyrimidin-7-onc, vardenafil or 10 tadalafil or a pharmaceutically acceptable sait or solvaté thereof, preferably sildenafilor a pharmaceutically acceptable sait or solvaté thereof.

As a yet further preferred aspect of the présent invention, the combination isselected from: 15 gabapentin and sildenafil; gabapentin and vardenafil;gabapentin and tadalafil; gabapentin and l-{6-ethoxy-5-[3-ethyl-6,7-dihydro-2-(2-methoxyethyl)-7-oxo-2H-pyrazolo[4,3- d]pyrmüdin-5-yl]-3-pyridylsulfonyl}-4-ethylpiperazine; 20 gabapentin and 5-(5-acetyl-2-butoxy-3-pyridinyl)-3-ethyl-2-(l-ethyl-3- -, azetidinyl)-2,6-dihydro-777-pyrazolo[4,3-<7]pyrimidin-7-one;pregabalin and sildenafil;pregabalin and vardenafil;pregabalin and tadalafil; 25 pregabalin and 1 - {6-ethoxy-5-[3-ethyl-6,7-dihydro-2-(2-methoxyethyl)-7-oxo- 2H-pyrazolo[4,3- d]pyrimidin-5-yl]-3-pyridylsulfonyl }-4-ethylpiperazine; pregabalin and 5-(5-acetyl-2-butoxy-3-pyridinyl)-3-ethyl-2-(l -ethyl-3-azetidinyl)-2,6-dihydro-72ï-pyrazolo[4,3-<ï]pyrimidin-7-one; [(lR,5R,6S)-6-(Aminomethyl)bicyclo[3.2.0]hept-6-yl]acetic acid, and30 sildenafil; [(lR,5R,6S)-6-(Aminomethyl)bicyclo[3,2.0]hept-6-yl]acetic acid, andvardenafil; 012899 24 .[(lR,5R,6S)-6-(Aminomethyl)bicyclo[3.2.0]hept-6-yl]acetic acid, andtadalafil; [(lR,5R,6S)-6-(Aminomethyl)bicyclo[3.2.0]hept-6-yl]acetic acid, and l-{6-ethoxy-5-[3-ethyl-6,7-dihydro-2-(2-methoxyethyl)-7-oxo-2H-pyrazolo[4,3- 5 d]pyrimidin-5-yl]-3-pyridylsulfonyl}-4-ethylpiperazine; and [(lR,5R,6S)-6-(Aimnomethyl)bicyclo[3.2.0]hept-6-yl]acetic acid, and 5-(5-acetyl-2-butoxy-3-pyridinyl)-3-ethyl-2-(l-ethyl-3-azetidinyl)-2,6-dihydro-777-pyrazolo[4,3-d]pyrimidin-7-one. (la,3a,5a)(3-amino-inethyl-bicyclo[3.2.0]hept-3-yl)-acetic acid, andi 10 sildenafil; (la,3a,5a,)(3-amino-methyl-bicyclo[3.2.0]hept-3-yl)-acetic acid, andvardenafil; (la,3a,5a)(3-amino-methyl-bicyclo[3.2.0]hept-3-yI)-acetic acid, and tadalafil; (la,3a,5a)(3-amino-methyl-bicyclo[3.2.0]hept-3-yl)-acetic acid, and l-{6-15 ethoxy-5-[3-ethyl-6,7-dihydro-2-(2-methoxyethyl)-7-oxo-2H-pyrazolo[4,3- d]pyrimidin-5-yl]-3-pyridylsulfonyl }-4-ethylpiperazine; and (la,3cc,5a)(3-amino-methyl-bicyclo[32.0]hept-3-yl)-acetic acid, and 5-(5-acetyl-2-butoxy-3-pyridinyl)-3-ethyl-2-(l-ethyl-3-azètidinyI)-2,6-dihydro-7/7-pyrazolo[4,3-d]pyrimidin-7-one; 20 (3S,4S)-(l-Aminomethyl-3,4-dimethyl-cyclopentyl)-acetic acid, and sildenafil; (35.45) -(l-Aminomethyl-3,4-dimethyl-cyclopentyl)-acetic acid, andvardenafil; (35.45) -(l-Aminometiiyl-3,4-dimethyl-cyclopentyl)-acetic acid, and tadalafil; 25 (3S,4S)-(l-Aminomethyl-3,4-dixnethyl-cyclopentyl)-acetic acid, and l-{ 6- ethoxy-5-[3-ethyl-6,7-dihydro-2-(2-methoxyethyl)-7-oxo-2H-pyrazolo[4,3- d]pyrimidin-5-yl]-3-pyridylsulfonyl}-4-ethylpiperazine, (35.45) -(l-Aminomethyl-3,4-dimethyl-cyclopentyl)-acetic acid, and 5-(5-acetyl-2-butoxy-3-pyridinyl)-3-ethyl-2-(l-ethyl-3-azetidinyl)-2,6-dihydro-7ff- 30 pyrazoIo[4,3-d]pyiimidin-7-one, or pharmaceutically acceptable salts or solvatés of any thereof.

012899 25

The combination of the présent invention in a single dosage fofm is suitablefor administration to any mammalian subject, preferably human. Administration maybe once (o.d.), twice (b.i.d.) or three times (t.i.d.) daily, suitably b.i.d. or t.i.d., moresuitably b.i.d, most suitably o.d.. Thus, as a further aspect of the présent invention, 5 there is provided a method of curative, prophylactic or palliative treatment of pain in amammalian subject comprising once, twice or thrice, suitably twice or thrice, moresuitably twice, most suitably once daily administration of an effective, particularlysynergistic, combination of an alpha-2-delta ligand and a PDEV inhibitor.

15 20

25

Determining a synergistic interaction between one or more components, theoptimum range for the effect and absolute dose ranges of each component for theeffect may be definitively measured by administration of the components overdifferent w/w ratio ranges and doses to patients in need of treatment. For humans, thecomplexity and cost of carrying out clinical studies on patients renders impractical theuse of this form of testing as a primary modeî for synergy. However, the observationof synergy in one species can be prédictive of the effect in other species and animalmodels exist, as described herein, to measure a synergistic effect and the results ofsuch studies can also be used to predict effective dose and plasma concentration ratioranges and the absolute doses and plasma concentrations required in other species bythe application of phaimacokinetic/pharmacodynamic methods. Establishedcorrélations between animal models and effects seen in man suggest that synergy inanimais is best-demonstrated using static and dynamic allodynia measurements inrodents that hâve undergone surgical (e.g. chronic constriction injury) or Chemical(e.g. streptozocin) procedures to induce the allodynia. Because of plateau effects insuch models, their value is best assessed in tenus of synergistic actions that inneuropathie pain patients would translate to dose-sparing advantages. Other modelsin which existing agents used for the treatment of neuropathie pain give only a partialresponse are more suited to predict the potential of combinations acting synergistically to produce increased maximal effîcacy at maximally tolerated doses ofthe two components. 30 26

Thus, as a further aspect of the présent invention, there is provided asynergistic combination for human administration comprising an alpha-2-delta ligandand a PDEV inhibitor, or pharmaceutically acceptable salts or solvatés thereof, in aw/w combination range which corresponds to the absolute ranges observed in a non- 5 human animal model, preferably a rat model, primarily used to identify a synergisticinteraction. Suitably, the ratio range in humans corresponds to a non-human rangeselected from between 1:50 to 50:1 parts by weight, 1:50 to 20:1,1:50 to 10:1,1:50 to1:1,1:20 to 50:1,1:20 to 20:1,1:20 to 10:1,1:20 to 1:1,1:10 to 50:1,1:10 to 20:1, 1:10 to 10:1, 1:10 to 1:1,1:1 to 50:1, 1.1 to 20:1 and 1:1 to 10:1. More suitably, the 10 human range corresponds to a synergistic non-human range of 1:10 to 20:1 parts byweight. Preferably, the human range corresponds to a non-human range of the orderof 1:1 to 10:1 parts by weight. For gabapentin and sildenafil, the human rangecorresponds to a synergistic dose range in a non-human, preferably rat, model of theorder of 1:1 to 10:1 parts by weight. 15

For humans, several experimental pain models may be used in man todemonstrate that agents with proven synergy in animais also hâve effects in mancompatible with that synergy. Examples of human models that may be fit for thispurpose include the heat/capsaicin model (Petersen, K.L. & Rowbotham, M.C. (1999) 20 NeuroReport 10,1511-1516), the i.d capsaicin model (Andersen, O.L., Felsby, S.,Nicolaisen, L., Bjemng, P., Jsesn, T.S. & Arendt-Nielsen, L. (1996) Pain 66, 51-62),including the use of repeated capsaicin trauma (Witting, N., Svesson, P., Arendt-Nielsen, L. &Jensen, T.S. (2000) Somatosensory Motor Res. 17, 5-12), andsummation or wind-up responses (Curatolo, M. et al. (2000) Anesthesiology 93, 1517 25 - 1530). With these models, subjective assessment of pain intensity or areas of hyperalgesia may be used as endpoints, or more objective endpoints, reliant onelectrophysiological or imaging technologies (such as functional magnetic résonanceimaging) may be employed (Bomhovd, K., Quante, M., Glauche, V., Bromm, B.,Weiller, C. & Buchel, C. (2002) Brain 125,1326-1336). Ail such models require 30 evidence of objective validation before it can be concluded that they provide evidencein man of supporting the synergistic actions of a combination that hâve been observedin animal studies. 012899 27

For the présent invention in humans, a suitable alpha-2-delta ligand:PDEVinhibitor ratio range is selected from between 1:50 to 50:1 parts by weight, 1:50 to20:1,1:50 to 10:1,1:50 to 1:1,1:20 to 50:1,1:20 to 20:1,1:20 to 10:1,1:20 to 1:1, 5 1:10 to 50:1,1:10 to 20:1,1:10 to 10:1,1:10 to 1:1,1:1 to 50:1,1.1 to 20:1 and 1:1 to 10:1, more suitably l:10to 20:1, preferably, 1:1 to 10:1. For a combination ofgabapentin and sildenafil, the invention provides a suitable dose in the ratio range of1:10 to 10:1 w/w, more suitably 1:5 to 5:1 respectively. L0 Optimal doses of each component for synergy can be determined according to published procedures in animal models. However, in man (even in experimentalmodels of pain) the cost can be very high for studies to détermine the entire exposure-response relationship at ail therapeutically relevant doses of each component of acombination. It may be necessary, at least initially, to estimate whether effects can be 15 observed that are consistent with synergy at doses that hâve been extrapolated fromthose that give optimal synergy in animais. In scaling the doses from animais to man, > factors such as relative body weight/body surface area, relative absorption,distribution, metabolism and excrétion of each component and relative plasmâ proteinbinding need to be considered and, for these reasons, the optimal dose ratio predicted 20 for man (and also for patients) is unlikely to be the same as the dose ratio shown to beoptimal in animais. However, the relationship between the two can be understood and ? calculated by one skilled in the art of animal and human pharmacokinetics. Importantin establishing the bridge between animal and human effects are the plasmaconcentrations obtained for each component used in the animal studies, as these are 25 related to the plasma concentration of each component that would be expected toprovide efficacy in man. Pharmacokinetic/ pharmacodynamie modeling (includingmethods such as isobolograms, interaction index and response surface modelling) andsimulations may help to predict synergistic dose ratios in man, particularly whereeither or both of these components has already been studied in man. 30

It is important to ascertain whether any concluded synergy observed inanimais or man is due solely to pharmacokinetic interactions. For example, inhibition 012899 28 of the metabolism of one compound by another xnight give a false impression ofpharmacodynamie synergy. In animal studies with gabapentin and sildenafil, repeatedblood samples hâve been taken and it has been shown that, in accordance with theknown pharmacokinetic properties of the agents, there is no evidence of any 5 pharmacokinetic interaction when the compounds are administered at the doses thatinduced synergistic pain interactions. This proves that the synergy with respect topain is pharmacodynamie, occurring subséquent to each of these agents interactingwith their respective receptor and/or enzyme targets. 10 Thus, according to a further aspect of the présent invention, there is provided a synergistic combination for administration to humans comprising an aIpha-2-deltaligand and a PDEV inhibitor or pharmaceutically acceptable salts or solvatés thereof,where the dose range of each component corresponds to the absolute synergisticranges observed in a non-human animal model, preferably the rat model, primarily 15 used to identify a synergistic interaction. Suitably, the dose range of alpha-2-delta ligand in human corresponds to a dose range of l-20mg/kg, more suitably l-10mg/kg,in the rat and the corresponding dose range for a PDEV inhibitor is 0.1-10mg/kg,more suitably 0.1-lmg/kg. For gabapentin and sildenafil, the dose range in the humansuitably corresponds to a synergistic range of l-10mg/kg gabapentin and 0.1-lmg/kg 20 sildenafil in the rat.

Suitably, the dose of alpha-2-delta ligand for use in a human is in a rangeselected from l-1200mg, l-500mg, l-100mg, l-50mg, l-25mg, 500-1200mg, 100-1200mg, 100-500mg, 50-1200mg, 50-500mg, or 50-100mg, suitably 50-100mg, b.i.d. 25 or t.i.d., suitably t.i.d., and the dose of PDEV inhibitor is in a range selected from 1-200mg„ l-100mg, l-50mg, l-25mg, 10-100mg, 10-50mg or 10-25 mg, suitably 10-lOOmg, b.i.d or t.i.d, suitably t.i.d.. For gabapentin and sildenafil, the suitable doseranges are 50-600mg:10-100nig t.i.d.. 30 It will be apparent to the skilled reader that the plasma concentration ranges of the alpha-2-delta ligand and PDEV inhibitor combinations of the présent inventionrequired to provide a therapeutic effect dépend on the species to be treated, and 012899 29 components used. For example, for gabapentin and sildenafil in the rat the Cmax values of gabapentin range from 0.520pg/ml to 10.5gg/ml and the Cmax values of sildenafil range from 0.02/xg/ml to 2. lgg/ml. 5 It is possible, using standard PK/PD and allometric methods, to extrapolate the plasma concentration values observed in an animal model to predict the values in adifferent species, particularly human. Thus, as a further aspect of the présentinvention, there is provided a synergisüc combination for administration to humanscomprising an alpha-2-delta ligand and aPDEV inhibitor, where the plasma 10 concentration range of each component corresponds to the absolute ranges observedin a non-human animal model, preferably the rat model, primarily used to identify asynergistic interaction. Suitably, the plasma concentration range in the humancorresponds to a range of 0.05/zg/ml to 10.5pg/ml for an alpha-2-delta ligand and0.005/zg/ml to 2.1/rg/ml for a PDEV inhibitor in the rat model. For gabapentin and 15 sildenafil, the plasma concentration range in the human corresponds to a range of0.05pg/ml to 10.5pg/ml for gabapentin and 0.005/ig/ml to 2.1pg/ml for sildenafil inthe rat model. Since protein-binding properties are similar in rat and human plasmafor both compounds, the plasma concentration ranges above are relevant to human. 20 Thus, an alternative aspect, the présent invention provides a synergistic combination comprising an alpha-2-delta ligand and a PDEV inhibitor, orphaimaceutically acceptable salts or solvatés thereof, where the plasma concentrationrange for the components comprises Cmax values of up to 20/xg/ml for the alpha-2-delta ligand and up to 4/xg/ml for a PDEV inhibitor, more suitably 0.5/xg/ml to 25 10/u.g/ml and 0.02/xg/ml to 2.1/xg/ml, preferably 0.05/xg/ml to 20/xg/ml and 0.005/ig/ml to 4/xg/ml respectively.

Particularly preferred combinations of the invention include those in whicheach variable of the combination is selected from the suitable parameters for each 30 variable. Even more préférable combinations of the invention include those whereeach variable of the combination is selected from the more suitable, most suitable,preferred or more preferred parameters for each variable. 012899 30

BRIEF DECRIPTIQN OF THE DRAWINGS

Figure 1. Effect of (a) gabapentin and (b) sildenafil on the maintenance of5 CCI-induced static ailodynià. Baseline (BL) paw withdrawal thresholds (PWT) tovon Frey hairs were determined in CCI animais before drug administration. PWTwere re-examined up to 4h post drug. Results are expressed as médian force (g)required to induce paw withdrawal (vertical bars represent lst and 3rd quartiles).*P<0.05 :,:*P<0.01 ***P<0.005 significantly different (Mann Wbitney U test) from 10 vehicle treated group at each time point.

Figure 2. Effect of (a) gabapentin and (b) sildenafil on the maintenance ofCCI-induced dynamic allodynia. Baseline (BL) paw withdrawal latencies (PWL) tocotton bud stimulus were determined for right hind paw before drug administration. 15 PWL’s were re-examined for up to 4 hours. Results are expressed as mean PWL (s)vertical bars represent ± SEM * P <0.05, ** P < 0.01, Significantly different(ANOVA followed by a Dunnett’s ί-test) from vehicle treated group at each timepoint. 20 Figure 3. Effect of fixed dose ratios of gabapentin and sildenafil on the maintenance of CCI-induced static allodynia. Ail data is expressed at the 2h timepoint post drug administration. Dose-response data for gabapentin and sildenafil alonewere taken from figure 1. Fixed dose ratios of. (a) 1:10 (b) 1:1 (c) 10:1 (d) 20:1gabapentin and sildenafil combinations. Results are expressed as médian force (g) 25 required to induce paw withdrawal (vertical bars represent lst and 3rd quartiles).

Figure 4. Effect of fixed dose ratios of gabapentin and sildenafil on themaintenance of CCI-induced dynamic allodynia. Ail data is expressed at the 2htime point post drug administration. Dose-response data for gabapentin and sildenafil 30 alone were taken from figure 2. Fixed dose ratios of (a) 1:10 (b) 1:1 (c) 10:1 (d) 20:1gabapentin and sildenafil combinations. Results are expressed as mean PWL (s) 31 vertical bars represent ± SEM *P <0.05, **P < 0.01, Significantiy different (ANOVAfollowed by a Dunnett’s ί-test) from vehicle treated group at each time point.

DETAILED DESCRIPTION QF THE INVENTION 5

The compounds of the présent combination invention can exist in unsolvatedforms as well as solvated forms, including hydrated forms. In general, the solvatedforms, including hydrated forms, which may contain isotopic substitutions (e.g. D2O,d6-acetone, d6-DMSO), are équivalent to unsolvated forms and are encompassed £^)10 within the scope of the présent invention.

Certain of the compounds of the présent invention possess one or more chiralcenters and each center may exist in the R or S configuration. The présent inventionincludes ail enantiomeric and epimeric forms as well as the appropriate mixtures 15 thereof. Séparation of diastereoisomers or cis and trans isomers may be achieved byconventional techniques, e.g. by fractional crystallisation, chromatography orH.P.L.C. of a stereoisomeric mixture of a compound of the invention or a suitable saitor dérivative thereof. 20 A number of alpha-2-delta ligands of the présent invention are amino acids.

Since amino acids are amphoteric, pharmacologically compatible salts can be salts ofappropriate non-toxic inorganic or organic acids or bases. Suitable acid addition saltsare the acetate, aspartate, benzoate, besylate, bicarbonate/carbonate, bisulphate,camsylate, citrate, edisylate, esylate, fumarate, gluceptate, gluconate, glucuronate, 25 hibenzate, hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide,hydrogen phosphate, isethionate, D- and L-lactate, malate, maleate, malonate,mesylate, methylsulphate, 2-napsylate, nicotinate, nitrate, orotate, palmoate,phosphate, saccharate, stéarate, succinate sulphate, D- and L-tartrate, and tosylatesalts. Suitable base salts are formed from bases which form non-toxic salts and 30 examples are the sodium, potassium, aluminium, calcium, magnésium, zinc, choline,diolamine, olamine, arginine, glycine, tromethamine, benzathine, lysine, meglumineand diethylamine salts. Salts with quatemary ammonium ions can also be prepared 012899 32 with, for example, the tetramethyl-ammonium ion. The compounds of the inventionmay also be formed as a zwitterion. Furthermore, since a number of the PDEVinhibitors of the présent invention are amines and a number of the alpha-2-deltaligands hâve an acid, functionality, a further aspect of the présent invention comprises 5 a sait form containing the 2 components, particulariy in a 1:1 combination. A suitable. combination sait form is the sait formed by a 1:1 combination of gabapentin and sildenafil. A suitable sait for amino acid compounds of the présent invention is the10 hydrochloride sait. For a review on suitable salts see Stahl and Wermuth, Handbook \ of Pharmaceutical Salts: Properties, Sélection, and Use, Wiley-VCH, Weinheim,

Germany (2002).

Also within the scope of the invention are clathrates, drug-host inclusion15 complexes wherein, in contrast to the aforementioned solvatés, the drug and host areprésent in non-stoichiometric amounts. For a review of such complexes, see J Pharm

Sci, 64 (8), 1269-1288 by Haleblian (August 1975).

Hereinafter ail references to compounds of the invention include references to20 salts thereof and to solvatés and clathrates of compounds of the invention and salts thereof.

Also included within the présent scope of the compounds of the invention arepolymorphe thereof. 25

Prodrugs of the above compounds of the invention are included in the scope ofthe instant invention. The chemically modified drug, or prodrug, should hâve adifferent pharmacokinetic profile to the parent, enabling easier absorption across themucosal epithelium, better sait formulation and/or solubility, improved systemic 30 stability (for an increase in plasma half-life, for example). These Chemicalmodifications may be 012899 (1) Ester or amide dérivatives which may be cleaved by, for example, esterases or lipases. For ester dérivatives, the ester is derived from the carboxylic acid moiety of the drug molécule by known means. For amide dérivatives, the amide may be derived from the carboxylic acid moiety or the amine moiety of the drug molécule by known means. 33 (2) Peptides which may be recognized by spécifie or nonspecificprotéinases. A peptide may be coupled to the drug molécule via amide bondformation with the amine or carboxylic acid moiety of the drug molécule byknown means.

(3) Dérivatives that accumulate at a site of action throughmembrane sélection of a prodrug form or modifiedprodrug form. (4) Any combination of 1 to 3.

Axninoacyl-glycolic and -lactic esters are known as prodrugs of amino acids 15 (Wermuth C.G., Chemistry and Industry, 1980:433-435). The carbonyl group of theamino acids can be esterified by known means. Prodrugs and soft drugs are known inthe art (Palomino E., Drugs of the Future, 1990;15(4):361-368). The last twocitations are hereby incorporated by reference. 20

The combination of the présent invention is useful for the general treatment of pain, particularly neuropathie pain. Physiological pain is an important protectivemechanism designed to wam of danger from potentially injurious stimuli from theextemal environment. The System opérâtes through a spécifie set of primary sensoryneurones and is exclusively activated by noxious stimuli via peripheral transducing 25 mechanisms (Millan 1999 Prog. Neurobio. 57: 1-164 for an intégrative Review).These sensory fibres are known as nociceptors and are characterised by smalldiameter axons with slow conduction velocities. Nociceptors encode the intensity,duration and quality of noxious stimulus and by virtue of their -topographicallyorganised projection to the spinal cord, the location of the stimulus. The nociceptors 30 are found on nociceptive nerve fibres of which there are two main types, A-deltafibres (myelinated) and C fibres (non-myelinated). The activity generated bynociceptor input is transferred after complex processing in the dorsal hom, either Û 12899 34 directly or via brain stem relay nuclei to the ventrobasal thalamus and then on to thecortex, where the sensation of pain is generated.

Intense acute pain and chronic pain may involve the same pathways driven by 5 pathophysiological processes and as such cease to provide a protective mechanismand instead contribute to debilitating symptoms associated with a wide range ofdisease States. Pain is a feature of many trauma and disease States. When a substantialinjury, via disease or trauma, to body tissue occurs the characteristics of nociceptoractivation are altered. There is sensitisation in the periphery, locally around the injury 10 and centrally where the nociceptors terminate. This leads to hypersensitivity at thesite of damage and in nearby normal tissue. In acute pain these mechanisms can beuseful and allow for the repair processes to take place and the hypersensitivity retumsto normal once the injury has healed. However, in many chronic pain States, thehypersensitivity far outlasts the healing process and is normally due to nervous System 15 injury. This injury often leads to maladaptation of the afferent fibres (Woolf & Salter2000 Science 288: 1765-1768). Clinical pain is présent when discomfort andabnormal sensitivity feature among the patient’s symptoms. Patients tend to be quiteheterogeneous and may présent with various pain symptoms. There are a number oftypical pain subtypes: 1) spontaneous pain which may be dull, buming, or stabbing; 2) 20 pain responses to noxious stimuli are exaggerated (hyperalgesia); 3) pain is producedby normally innocuous stimuli (allodynia) (Meyer et al., 1994 Textbook of Pain 13-44). Although patients with back pain, arthritis pain, CNS trauma, or neuropathiepain may hâve similar symptoms, the underlying mechanisms are different and,therefore, may require different treatment strategies. Therefore pain can be divided 25 into a number of different areas because of differing pathophysiology, these includenociceptive, inflammatory, neuropathie pain etc. It should be noted that some typesof pain hâve multiple aetiologies and thus can be classified in more than one area, e.g.Back pain, Cancer pain can hâve nociceptive inflammatory and neuropathiecomponents, 30

Nociceptive pain is induced by tissue injury or by intense stimuli with thepotential to cause injury. Pain afferents are activated by transduction of stimuli by 012899 35 nociceptors at the site of injury and sensitise the spinal cord at the level of theirtermination, This is then relayed up the spinal tracts to the brain where pain isperceived (Meyer et al., 1994 Textbook of Pain 13-44). The activation of nociceptorsactivâtes two types of afferent nerve fibres. Myelinated A-delta fibres transmitted 5 rapidly and are responsible for the sharp and stabbing pain sensations, whilstunmyelinated C fibres transmit at a slower rate and convey the dull or aching pain.Moderate to severe acute nociceptive pain is a prominent feature of, but is not limitedto pain from strains/sprains, post-operative pain (pain foliowing any type of surgicalprocedure), posttrauxnatic pain, bums, myocardial infarction, acute pancreatitis, andrfjgiO rénal colic. Also cancer related acute pain syndromes commonly due to therapeuticinteractions such as chemotherapy toxicity, immunotherapy, hormonal therapy andradiotherapy. Moderate to severe acute nociceptive pain is a prominent feature of, butis not limited to, cancer pain which may be tumour related pain, (e.g. bone pain,headache and facial pain, viscera pain) or associated with cancer therapy (e.g. 15 postchemotherapy syndromes, chronic postsurgical pain syndromes, post radiationsyndromes), back pain which may be due to hemiated or ruptured intervertébral disesor abnormalities of the lumber facet joints, sacroiliac joints, paraspinal muscles or theposterior longitudinal ligament 20 Neuropathie pain is defined as pain initiated or caused by a primary lésion or dysfunction in the nervous System (IASP définition). Nerve damage can be caused by — tourna and disease and thus the term ‘neuropathie pain’ encompasses many disorderswith diverse aetiologies. These include but are not limited to, diabetic neuropathy,post herpetic neuralgia, back pain, cancer neuropathy, chemotherapy-induced 25 neuropathy, HTV neuropathy, Phantom limb pain, Carpal Tunnel Syndrome, chronicalcoholism, hypothyroidism, trigeminal neuralgia, uremia, trauma-inducedneuropathy, or vitamin deficiencies. Neuropathie pain is pathological as it has noprotective rôle. It is often présent well after the original cause, has dissipated,commonly lasting for years, significantly decreasing a patients quality of life (Woolf 30 and Mannion 1999 Lancet 353: 1959-1964). The symptoms of neuropathie pain aredifficult to treat, as they are often heterogeneous even between patients with the samedisease (Woolf & Decosterd 1999 Pain Supp. 6: S141-S147; Woolf and Mannion 01289 36 1999 Lancet 353: 1959-1964). They include spontaneous pain, which can becontinuons, orparoxysmal and abnormal evoked pain, such as hyperalgesia (increasedsensitivity to a noxious stimulus) and allodynia (sensitivity to a normally innocuousstimulus). 5

The inflammatory process is a complex sériés of biochemical and cellularevents activated in response to tissue injury or the presence of foreign substances,which resuit in swelling and pain (Levine and Taiwo 1994: Textbook of Pain 45-56).Arthritic pain makes up the majority of the inflammatory pain population. 10 Rheumatoid disease is one of the commonest chronic inflammatory conditions indeveloped countries and rheumatoid arthritis (RA) is a common cause of disability.The exact aetiology of RA is unknown, but current hypothèses suggest that bothgenetic and microbiological factors may be important (Grennan & Jayson 1994Textbook of Pain 397-407). It has been estimated that almost 16 million Americans 15 hâve symptomatic osteoarthritis (OA) or degenerative joint disease, most of whom areover 60 years of âge, and this is expected to increase to 40 million as the âge of thepopulation increases, making this a public health problem of enormous magnitude(Houge & Mersfelder 2002 Ann Pharmacother. 36: 679-686; McCarthy et al., 1994Textbook of Pain 387-395). Most patients with OA seek medical attention because of 20 pain. Arthritis has a significant impact on psychosocial and physical fraction and isknown to be the Ieading cause of disability in later life. Other types of inflammatorypain include but are not limited to inflammatory bowel diseases (IBD),

Other types of pain include but are not limited to; - Musculo-skeletal disorders includirtg but not limited to myalgia,fibromyalgia, spondylitis, sero-negative (non-rheumatoid) arthropathies, non-articularrheumatism, dystrophinopathy, Glycogenolysis, polymyositis, pyomyositis. 30 - Central pain or ‘thalamic pain’ as defined by pain caused by lésion or dysfunction of the nervous system including but not limited to central post-strokepain, multiple sclerosis, spinal cord injury, Parkinson’s disease and epilepsy. 37

X - Heart and vascular pain including but not Iimited to angina, myocardica]infarction, mitral stenosis, pericarditis, Raynaud’s phenomenon, sclerodoma, skeletalmuscle ischemia. 5 - Viscéral pain, and gastrointestinal disorders. The viscera encompasses theorgans of the abdominal cavity. These organs include the sex organs, spleen and partof the digestive System. Pain associated with the viscera may be neuropathie,nociceptive as well as inflammatory and can be divided into digestive viscéral pain .0 and non-digestive viscéral pain. Commonly encountered gastrointestinal (GI)disorders include the functional bowel disorders (FBD) and the inflammatory boweldiseases (IBD). These GI disorders include a wide range of disease States that arecurrently only moderately controlled, including - for FBD, gastro-esophageal reflux,dyspepsia, the irritable bowel syndrome (IBS) and functional abdominal pain 15 syndrome (FAPS), and - for IBD, Crohn’s disease, ileitis, and ulcerative colitis,which ail regularly produce viscéral pain. Other types of viscéral pain include the painassociated with dysmenorrhea, pelvic pain, cystitis and pancreatitis. - Head pain including but not Iimited to migraine, migraine with aura, 20 migraine without aura, cluster headache, tension-type headache. î - Orofacial pain including but not Iimited to dental pain, temporomandibularmyofascial pain. 25 Thus, as a yet further aspect, there is provided the simultaneous, sequential or separate use of an alpha-2-delta ligand, excluding compounds of formula (i)-(xxv) ofPCT/1B02/01146 and pregabalin or gabapentin, where the exclusion of pregabalin orgabapentin is Iimited to use in the treatment of neuropathy, and a PDEV inhibitor inthe manufacture of a médicament for the curative, prophylactic or palliative treatment 30 of pain, particularly neuropathie pain. As a preferred feature, the use suitablycomprises any one of the combinations mentioned herein above. 012899 38

As an alternative aspect, there is provided a method for the curative,prophylactic or palliative treatment of pain, particularly neuropathie pain, comprisingsimultaneous, sequential or separate administration of a therapeutically effectiveamount of an alpha-2-delta ligand, excluding pregabalin or gabapentin, where the 5 exclusion of pregabalin or gabapentin is limited to use in the treatment of neuropathy,and a PDEV inhibitor to a mammal in need of said treatment. As an alternativeaspect, the exclusion may the include compounds of formula (i)-(xxv) ofPCT/1B02/01146. As a preferred feature, the method suitably comprises any one ofthe combinations mentioned herein above. 10

As an alternative aspect, there is provided the simultaneous, sequential orseparate use of a synergistic combination of an alpha-2-delta ligand and a PDEVinhibitor in the manufacture of a médicament for the curative, prophylactic orpalliative treatment of pain, particularly neuropathie pain. As a preferred feature, the 15 use suitably comprises any one of the combinations mentioned herein above.

As a further alternative aspect, there is provided a method for the curative,prophylactic or palliative treatment of pain, particularly neuropathie pain, comprisingsimultaneous,. sequential or separate administration of a therapeutically synergistic 20 amount of an alpha-2-delta ligand and a PDEV inhibitor to a mammal in need of saidtreatment. As a preferred feature, the method suitably comprises any one of thecombinations mentioned herein above.

The biological activity of the alpha-2-delta ligands of the invention may be 25 measured in a radioligand binding assay using [3H]gabapentin and the α2δ subunit derived from porcine brain tissue (GeeN.S., Brown J.P., Dissanayake V.U.K.,Offord J., Thurlow R., Woodruff G.N., J. Biol. Chem., 1996;271:5879-5776). Resultsmay be expressed in terms of μΜ or nM α2δ binding affinity. 30 In vitro inhibitory activities of the PDEV inhibitors of the présent invention against cyclic guanosine monophosphate (cGMP) may be determined bymeasurement of their IC50 values, according to the details described in WOOl/27113.

39

Functional activity can be assessed as described by SA Ballard et al (Brit. J.

Pharmacology, 1996,118 (suppl.), abstract 153P).

The éléments of the combination of the instant invention may be administered5 separately, simultaneously or sequentially. As a further aspect of the présentinvention, there is provided a package comprising a synergistic combination of an alpha-2-delta ligand and a PDEV inhibitor and a suitable container.

The combination of the présent invention may also optionally be administeredjLO with one or more other pharmacologically active agents. Suitable optional agents include: 15 20 25 30 (i) opioid analgésies, e.g. morphine, heroin, hydromorphone,oxymorphone, levorphanol, levallorphan, methadone, meperidine, fentanyl, cocaïne,codeine, dihydrocodéine, oxycodone, hydrocodone, propoxyphene, nalmefene,nalorphine, buprénorphine, butorphanol, nalbuphine and pentazocine; (ii) Opioid antagonists, e.g. naloxone, naltrexone (iii) nonsteroidal antiinflammatory drugs (NSAIDs), e.g. aspirin,diclofenac, difluinsal, etodolac, fenbufen, fenoprofen, flufenisal,flurbiprofenjbuprofen, indomethacin, ketoprofen, ketorolac, meclofenamic acid,mefenamic acid, nabumetone, naproxen, oxaprozin, phenylbutazone, piroxicam,sulindac, tolmetin, zomepirac, and their pharmaceutically acceptable salts or solvatés; (iv) barbiturate sédatives, e.g. amobarbital, aprobarbital,butabarbital, butabital, mephobarbital, metharbital, methohexital, pentobarbital,phenobartital, secobarbital, talbutal, theamylal, thiopental and their pharmaceuticallyacceptable salts or solvatés; (v) benzodiazépines having a sédative action, e.g.chlordiazepoxide, clorazepate, diazepam, flurazepam, lorazépam, oxazepam,temazepam, triazolam and their pharmaceutically acceptable salts or solvatés, (vi) Hi antagonists having a sédative action, e.g. diphenhydramine,pyrilamine, promethazine, chlorpheniramine, chlorcyclizine and theirpharmaceutically acceptable salts or solvatés; 012899 40 (vii) miscellaneous sédatives such as glutethimide, meprobamate,methaqualone, dichloralphenazone and their pharmaceutically acceptable salts orsolvatés; (viii) skeletal muscle relaxants, e.g. baclofen, tolperisone, 5 carisoprodol, chlorzoxazone, cyclobenzaprine, methocarbamol, orphrenadine and their pharmaceutically acceptable salts or solvatés, (ix) NMDA receptor antagoniste, e.g. dextromethorphan ((+)-3-hydroxy-N-methylmorphinan) and its métabolite dextrorphan ((+)-3-hydroxy-N-methylmorphinan), ketamine, memantine, pyrroloquinoline quinone and cis-4- 10 (phosphonomethyl)-2- piperidinecarboxylic acid and their pharmaceuticallyacceptable salts or solvatés; (x) alpha-adrenergic active compounds, e.g. doxazosin, tamsulosin,clonidine and 4-amino-6,7-dimethoxy-2-(5-methanesulfonamido-l, 2,3,4-tetrahydroisoquinol-2-yl)-5-(2-pyridyl) quinazoline; 15 (xi) tricyclic antidepressants, e.g. desipramine, imipramine, amytriptiline and nortriptiline; (xii) anticonvulsants, e.g. carbamazepine, valproate, lamotrigine; (xiii) serotonin reuptake inhibitors, e.g. fluoxetine, paroxetine,citalopram and sertraline; 20 (xiv) mixed serotonin-noradrenaline reuptake inhibitors, e.g. milnacipran, venlafaxine and duloxetine; (xv) noradrenaline reuptake inhibitors, e.g. reboxetine; (xvi) Tachykinin (NK) antagonists, particularly Nk-3, NK-2 and NK-1 antagonists e.g., (aR,9R)-7-[3,5-bis(trifluoromethyl)benzyl]-8,9,10,ll-tetrahydro- 25 9-methyl-5-(4-methylphenyl)-7H-[l,4]diazocino[2,l-g][l,7]naphthridine-6-13-dione(TAK-637), 5-[[(2R,3S)-2-[(lR)-l-[3,5-bis(trifluoromethyl)phenyI]ethoxy-3-(4-fluorophenyl)-4-morpholinyl]methyl]-l,2-dihydro-3H-l,2,4-triazol-3-one (MK-869),lanepitant, dapitant and 3-[[2-methoxy-5-(trifluoromethoxy)phenyl]methylamino]-2-phenyl-piperidine (2S,3S) 30 (xvii) Muscarinic antagonists, e.g oxybutin, tolterodine, propiverine, tropsium chloride and darifenacin; (xviii) COX-2 inhibitors, e.g. celecoxib, rofecoxib and valdecoxib; 012899 41 1 (xix) Non-selective COX inhibitors (preferably with GI protection),e.g. nitroflurbiprofen (HCT-1026); (xx) coal-tar analgésies, in particular, paracétamol; (xxi) neuroleptics, such as droperidol; 5 (xxii) Vanilloid receptor agonists, e.g. resinferatoxin; (xxiii) Beta-adrenergic compounds such as propranolol; (xxiv) Local anaesthetics, such as mexiletine, lidocaine; (xxv) Corticosteriods, such as dexamethasone(xxvi) serotonin receptor agonists and antagonists; , ,0 (xxvii) cholinergic (nicotinic) analgésies; and (xxviii)miscellaneous agents such as Tramadol®.

Thus, the présent invention extends to a combination product comprising analpha-2-delta ligand, a PDEV inhibitor, and one or more other therapeutic agents, 15 such as one of those listed above, for simultaneous, separate or sequential use in thecurative, prophylactic or palliative treatment of pain, particularly neuropathie pain.

The combination of the invention can be administered alone but one or bothéléments will generally be administered in an admixture with suitable pharmaceutical 20 excipient(s), diluent(s) or carrier(s) selected with regard to the intended route of„ v administration and standard pharmaceutical practice. If appropriate, auxiliaires can be added. Auxiliaries are preservatives, anti-oxidants, flavours or colourants. Thecompounds of the invention may be of immédiate-, delayed-, modified-, sustained-,pulsed- or controlled-release type. 25

The éléments of the combination of the présent invention can be administered,for example but not limited to, the following route: orally, buccally or sublingually inthe form of tablets, capsules, multi-and nano-particulates, gels, films (incl. muco-adhesive), powder, ovules, élixirs, lozenges (incl. liquid-filled), chews, solutions, 30 suspensions and sprays. The compounds of the invention may also be administered asosmotic dosage form, or in the form of a high energy dispersion or as coated particlesor fast-dissolving, fast -disintegrating dosage form as described in Ashley 01289 42

Publications, 2001 by Liang and Chen. The compounds of the invention may beadministered as crystalline or amorphous products, freeze dried or spray dried.Suitable formulations of the compounds of the invention may be in hydrophilic orhydrophobie matrix, ion-exchange resin complex, coated or uncoated form and other 5 types as described in US 6,106,864 as desired. Such pharmaceutical compositions, for example, tablets, may contain excipients such as microcrystallinecellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate,glycine and starch (preferably corn, potato or tapioca starch), mannitol, disintegrantssuch as sodium starch glycolate, crosscarmellose sodium and certain complex 10 silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), triglycérides, hydroxypropylcellulose(HPC), bentonite sucrose, sorbitol, gelatin and acacia. Additionally, lubricatingagents may be added to solid compositions such as magnésium stéarate, stearic acid,glyceryl behenate, PEG and talc or wetting agents, such as sodium lauryl sulphate. 15 Additionally, polymère such as carbohydrates, phospoholipids and proteins may beincluded.

Fast dispersing or dissolving dosage fromulations (FDDFs) may contain thefollowing ingrédients: aspartame, acesulfame potassium, citric acid, croscarmellose 20 sodium, crospovidone, diascorbic acid, ethyl acrylate, ethyl cellulose, gelatin, hydroxypropylmetbyl cellulose, magnésium stéarate, mannitol, methyl méthacrylate,mint flavouring, polyethylene glycol, fumed silica, Silicon dioxide, sodium starch, glycohate, sodium stearyl fumarate, sorbitol or xylitol. The ternis dispersing or „dissolving as used herein to describe FDDFs are dépendent upon the solubility of the 25 drug substance used, i.e. where the drug substance is insoluble a fast dispersing dosage form can be prepared and where the drug substance is soluble a fast dissolvingdosage form can be prepared.

The solid dosage form, such as tablets are manufactured by a standard process,30 for example, direct compression or a wet, dry or melt granulation, melt congealing and extrusion process. The tablet cores which may be mono or multi-layer may becoated with appropriate overcoats known in the art. 43 012899

Solid compositions of a similar type may also be employed as fillers in capsules such as gelatin, starch or HPMC capsules. Preferred excipients in this regard include lactose, starch, a cellulose, milk sugar or high molecular weight polyethylene 5 glycols. Liquid compositions may be employed as fillers in soft or hard capsules suchas gelatin capsule. For aqueous and oily suspensions, solutions, syrups and/or élixirs,the compounds of the invention may be combined with various sweetening orflavouring agents, colouring matter or dyes, with emulsifying and/or suspendingagents and with diluents such as water, éthanol, propylene glycol, methylcellulose, 10 alginic acid or sodium alginate, glycerin, oils, hydrocolloid agents and combinationsthereof. Moreover, formulations containing these compounds and excipients may be ’ presented as a dry product for constitution with water or other suitable vehicles beforeuse. 15 Liquid form préparations include solutions, suspensions, and émulsions, for example, water or water propylene glycol solutions. For parentéral injection, liquidpréparations can be formulated in solution in aqueous polyethylene glycol solution.Aqueous solutions suitable for oral use can be prepared by dissolving the activecomponent in water and adding suitable colorants, flavors, stabilizing and thickening 20 agents as desired. Aqueous suspensions suitable for oral use can be made bydispersing the finely divided active component in water with viscous materiel, such as ) natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose,and other well-known suspending agents. 25 The éléments of the combination of the présent invention can also be administered by injection, that is, intravenously, intramuscularly, intracutaneously,intraduodenally, or intraperitoneally, intraarterially, intrathecally, intraventricularly,intraurethrally, intrastemally, intracranially, intraspinally or subcutançously, or theymay be administered by infusion, needle-free injectors or implant injection 30 techniques. For such parentéral administration they are best used in the form of astérile aqueous solution, suspension or émulsion (or System so that can includemicelles) which may contain other substances known in the art, for example, enough 012899 44 salts or carbohydrates such as glucose to make the solution isotonie with blood. Theaqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), ifnecessary. For some forms of parentéral administration they may be used in the formof a stérile non-aqueous System such as fixed oils, including mono- or diglycerides, 5 and fatty acids, including oleic acid. The préparation of suitable parentéralformulations under stérile conditions for example lyophilisation is readilyaccomplished by standard pharmaceutical techniques well-known to those skilled inthe art. Altematively, the active ingrédient may be in pôwder form for constitutionwith a suitable vehicle (e.g. stérile, pyrogen-free water) before use. 10

Also, the éléments of the combination of the présent invention can beadministered intranasally or by inhalation. They are conveniently delivered in theform of a dry powder (either alone, as a mixture, for example a dry blend with lactose,or a mixed component particle, for example with phospholipids) from a dry powder 15 inhaler or an aérosol spray présentation from a pressurised container, pump, spray,atomiser (preferably an atomiser using electrohydrodynamics to produce a fine mist)or nebüliser, with or without the use of a suitable propellant, e.g.dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, ahydrofluoroalkane such as 1,1,1,2-tetrafluoroethane (HFA 134A [trade mark]) or 20 1,1,1,2,3,3,3-heptafluoropropane (HFA 227EA [trade mark]), carbon dioxide, afurther perfluorinated hydrocarbon such as Perflubron (trade mark) or other suitablegas. In the case of a pressurised aérosol, the dosage unit may be determined byproviding a valve to deîiver a metered amount. The pressurised container, pump,spray, atomiser or nebüliser may contain a solution or suspension of the active 25 compound, e.g. using a mixture of éthanol (optionally, aqueous éthanol) or a suitableagent for dispersing, solubilising or èxtending release and the propellant as thesolvent, which may additionally contain a lubricant, e.g. sorbitan trioleate. Capsules,blisters and cartridges (made, for example, from gelatin or HPMC). for use in aninhaler or insufflator may be formulated to contain a powder mix of the compound of 30 the invention, a suitable powder base such as lactose or starch and a performancemodifier such as 1-leucine, mannitol or magnésium stéarate.

012899 45 1

Prior to use in a dry powder formulation or suspension formulation forinhalation the éléments of the combination of the invention will be micronised to asize suitable for delivery by inhalation (typically considered as less than 5 microns).Micronisation could be achieved by a range of methods, for example spiral jet 5 milling, fluid bed jet milling, use of supercritical fluid crystallisation or by spraydrying. A suitable solution formulation for use in an atomiser usingelectrohydrodynamics to produce a fine mist may contain from lpg to lOmg of the LO compound of the invention per actuation and the actuation volume may vary from 1 toΙΟΟμΙ. A typical formulation may comprise a compound of the invention, propyleneglycol, stérile water, éthanol and sodium chloride. Alternative solvents may be usedin place of propylene glycol, for example glycerol or polyethylene glycol. 15 Altematively, the éléments of the combination of the invention may be administered topically to the skin, mucosa, dermally or transdermally, for example, inthe form of a gel, hydrogel, lotion, solution, cream, ointment, dusting powder,dressing, foam, film, skin patch, wafers, implant, sponges, fibres, bandage,microemulsions and combinations thereof. For such applications, the compounds of 20 the invention can be suspended or dissolved in, for example, a mixture with one ormore of the following: minerai oil, liquid petrolatum, white petrolatum, propyleneglycol, polyoxyethylene polyoxypropylene compound, emulsifying wax , fîxed oils,including synthetic mono- or diglycerides, and fatty acids, including oleic acid, water,sorbitan monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, cetyl 25 esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, alcohols such as éthanol. Altematively, pénétration enhancers may be used. The following may alsobe used polymers, carbohydrates, proteins, phospolipids in the form of nanoparticles(such as niosomes or liposomes) or suspended or dissolved. ht addition, they may bedelivered using iontophoresis, electroporation, phonophoresis and sonophoresis. 30

Altematively, the éléments of the combination of the invention can beadministered rectally, for example in the form of a suppository or pessary. They may

46 also be administered by vaginal route. For example, these compositions may beprepared by mixing the drug with a suitable non-irritant excipients, such as cocoabutter, synthetic glyceride esters or polyethylene glycols, which are solid at ordinarytempératures, but liquefy and/or dissolve in the cavity to release the drug. 5

The éléments of the combination of the invention may also be administered bythe ocular route. For ophthalmic use, the compounds can be formulated as micronisedsuspensions in isotonie, pH adjusted, stérile saline, or, preferably, as solutions inisotonie, pH adjusted, stérile saline. A polymer may be added such as crossed-linked 10 polyacrylic acid, polyvinylalcohol, hyaluronic acid, a cellulosic polymer (e.g.hydroxypropylmethylcellulose, hydroxyethylcellulose, methyl cellulose), or aheteropolysaccharide polymer (e.g. gelan gum). Altematively, they may beformulated in an ointment such as petrolatum or minerai oil, incorporated into bio-dégradable (e.g. absorbable gel sponges, collagen) or non-biodegradable (e.g. 15 silicone) implants, wafers, drops, lenses or delivered via particulate or vesicular

Systems such as niosomes or liposomes. Formulations may be optionally combinedwith a preservative, such as benzalkonium chloride. In addition, they may bedelivered using iontophoresis. They may also be administered in the ear, using forexample but not limited to the drops. 20

The éléments of the combination of the invention may also be used incombination with a cyclodextrin. Cyclodextrins are known to form inclusion and non-inclusion complexes with drug molécules. Formation of a drug-cyclodextrin complexmay modify the solubility, dissolution rate, taste-masking, bioavailability and/or 25 stability property of a drug molécule. Drug-cyclodextrin complexes are generallyuseful for most dosage forms and administration routes. As an alternative to directcomplexation with the drug the cyclodextrin may be used as an auxiliary additive, e.g.as a carrier, diluent or solubiliser. Alpha-, beta- and gamma-cyclodextrins are mostcommonly used and suitable examples are described in WO-A-91/11172, WO-A- 30 94/0251S and WO-A-98/55148. 012899 47

The terni ‘administered’ includes delivery by viral or non-viral techniques.Viral delivery mechanisms include but are not limited to adénoviral vectors, adeno-associated viral (AAV) vectors, herpes viral vectors, retroviral vectors, lentiviralvectors, and baculoviral vectors. Non-viral delivery mechanisms include lipid 5 mediated transfection, lipsomes, immunoliposomes, lipofectin, cationic facialamphiphiles (CFAs) and combinations thereof. The routes for such deliverymechanisms include but are not limited to mucosal, nasal, oral, parentéral,gastrointestinal, topical or sublingual routes.

15

Thus, as a further aspect of the présent invention, there is provided apharmaceutical composition comprising a combination comprising an alpha-2-deltaligand, excluding gabapentin, pregabalin, a PDEV inhibitor and a suitable excipient,diluent or carrier. Altematively, the exclusion may include the compounds of formula(i)-(xxv) of PCT/IB02/01146. Suitably, the composition is suitable for use in thetreatment of pain, particularly neuropathie pain.

As an alternative aspect of the présent invention, there is provided apharmaceutical composition comprising a synergistic combination comprising analpha-2-delta ligand, a PDEV inhibitor and a suitable excipient, diluent or carrier. 20 Suitably, the composition is suitable for use in the treatment of pain, particularlyneuropathie pain. ! y

For non-human animal administration the teim ‘pharmaceutical’ as usedherein may be replaced bÿ ‘ veterinary’. 25

The element of the pharmaceutical préparation is preferably in unit dosageform. In such form the préparation is subdivided into unit doses containingappropriate quantities of the active component. The unit dosage Jorm can be apackaged préparation, the package containing discrète quantities of préparation, such 30 as packeted tablets, capsules, and powders in vials or ampoules. Also, the unit dosageform can be a capsules, tablet, cachet, or lozenge itself, or it can be the appropriatenumber of any of these in packaged form. The quantity of active component in a unit

48 dose préparation may be varied or adjusted according to the particular application andthe potency of the active components. Generally, treatment is initiated with smallerdosages which are less than the optimum dose of the compounds. Thereafter, thedosage is increased by small incréments until the optimum effect under the 5 circumstances is reached. For convenience, the total daily dosage may be divided andadministered in portions during the day, if desired.

For veterinary use, a combination according o the présent invention orveterinarily acceptable salts or solvatés thereof, is administered as a suitably 10 acceptable formulation in accordance with normal veterinary practice and the veterinary surgeon will détermine the dosing regimen and route of administrationwhich will be most appropriate for a particular animal.

15 BIOLQGY EXAMFLES

METHODS

Animais 20 Male Sprague Dawley rats (200-250g), obtained from Charles River, (Margate, Kent, U.K.) were housed in groups of 6. Ail animais were kept under a 12hlight/dark cycle (lights on at 07h 00min) with food and water ad libitum. AUexperiments were canied out by an observer unaware of drug treatments. 25 CCI surgery in the rat

Animais were anaesthetised with isoflurane. The sciatic nerve was ligated aspreviously described by Bennett and Xie, 1988. Animais were placed on ahoméothermie blanket for the duration of the procedure. After surgical préparation thecommon sciatic nerve was exposed at the middle of the thigh by blunt dissection through 30 biceps femoris. Proximal to the sciatic trifurcation, about 7mm of nerve was freed ofadhering tissue and 4 ligatures (4-0 silk) were tied loosely around it with about 1mmspacing. The incision was closed in layers and the wound treated with topical antibiotics. 012899 49 \

Effect of combinations on the maintenance of CCI-induced static anddynamic allodynia

Dose-responses to gabapentin and sildenafil were first performed alone in the 5 CCI model. Combinations were examined following a fixed ratio design. A dose-response to each fixed dose ratio of the combination was performed. On each test day,baseline paw withdrawal thresholds (PWT) to von Frey hairs and paw withdrawallatencies (PWL) to a cotton bud stimulus were determined prior to drug treatment.Gabapentin was administered p.o. directly followed by s.c. administration of sildenafil p and PWT and PWL re-examined for up to 5h. The data are expressed at the 2h timepoint for both the static and dynamic data as this timepoint represent the peakantiallodynic effects.

Evaluation of allodynia 15 Static allodynia was measured using Semmes-Weinstein von Frey hairs (Stoelting, Illinois, U.S.A.). Animais were placed into wire mesh bottom cagesallowing access to the underside of their paws. Animais were habituated to thisenvironment prior to the start of the experiment. Static allodynia was tésted bytouching the plantar surface of the animais right hind paw with von Frey hairs in 20 ascending order of force ( 0.7, 1.2, 1.5, 2, 3.6, 5.5, 8.5, 11.8, 15.1 and 29g) for up to6sec. Once a withdrawal response was established, the paw was re-tested, startingwith the next descending von Frey hair until no response occurred. The highest forceof 29g lifted the paw as well as eliciting a response, thus represented the eut off point.The lowest amount of force required to elicit a response was recorded as the PWT in 25 grams.

Dynamic allodynia was assessed by lightly stroking the plantar suiface of thehind paw with a cotton bud. Care was taken to perform this procedure jn fullyhabituated rats that were not active to avoid recording general motor activity. At least 30 three measurements were taken at each time point the mean of which represented thepaw withdrawal latency (PWL). If no reaction was exhibited within 15s the procedurewas terminated and animais were assigned this withdrawal time. Thus 15s effectively 012899 représente no withdrawal. A withdrawal response Was often accompanied with repeated flinching or licking of the paw. Dynamic allodynia was considered to be présent if animais responded to the cotton stimulus before 8s of stroking.

5 REStLTS 50

Effect of gabapentin and sildenafil alone on CCI-induced static anddynamic allodynia

Gabapentin dose-dependently (10-100mg/kg, p.o.) blocked the maintenance10 of both static and dynamic allodynia with a minimum effective dose (MED) of lOmg/kg (Figure 1, 2). The dose of lOOmg/kg produced a complété blockade of theseresponses. Sildenafil dose-dependently (10-30mg/kg s.c.) blocked the maintenance ofstatic allodynia with a minimum effective dose of lOmg/kg and the dose of 30mg/kgproducing an approximate 60% blockade (Figure 1). Sildenafil had amodest effect on 15 the maintenance of dynamic allodynia with an MED of 30mgZkg producing a 25%blockade (Figure 2).

Effect of combinations of gabapentin and sildenafil on CCI-induced staticallodynia 20 Gabapentin and sildenafil had peak antiallodynic actions at 2h post administration in the CCI-induced static model. Thus, for clarity ail combination dataare expressed at this time point. Gabapentin and sildenafil were administered at fixeddose ratios of 1:10,1:1,10:1 and 20:1. Following fixed dose ratios of 1:10 and 20:1,combinations of gabapentin and sildenafil produced an additive interaction (Figure 3). 25 However, the fixed dose ratio of 1:1 and 10:1 demonstrated synergy with static allodynia completely blocked by a total dose of 20mg/kg and llmg/kg respectively(Figure 3). The 1:1 combination représente a 10-fold lower dose of gabapentin and 3-fold lower dose of sildenafil when administered alone whilst the 1:1 ratio représente a 10-fold lower dose of gabapentin and 30-fold lower dose of sildenafil when 30 administered alone. 51

Effect of combinations of gabapentin and sildenafil on CCI-induceddvnamic allodynia

Gabapentin and sildenafil had peak antiallodynic actions at 2h postadministration in the CCI-induced dynamic model. Thus, for clarity ail combination 5 data are expressed at this time point. Gabapentin and sildenafil were administered atfixed dose ratios of 1:10,1:1,10:1 and 20:1. Similar data were seen on dynamicallodynia to those with static allodynia. Following fixed dose ratios of 1:10 and 20:1,combinations of gabapentin and sildenafil produced an additive interaction (Figure 4).However, the fixed dose ratio of 1:1 and 10:1 demonstrated synergy with static ^10 allodynia completely blocked by a total dose of 20mg/kg and 1 lmg/kg respectively.The 1:1 combination represents a 10-fold lower dose of gabapentin and 3-fold lower ' dose of sildenafil when administered alone whilst the 1:1 ratio represents a 10-foldlower dose of gabapentin and 30-fold lower dose of sildenafil when administeredalone. 15

Similar experiments were also performed in the same model for a furtheralpha-2-delta ligand (pregabalin) in combination with sildenafil and also with-gabapentin and a further PDEV inhibitor, 3-Ethyl-5-[5-(4-ethyl-piperazine-l--sulfonyl)-2-propoxy-phenyl]-2-pyridin-2-ylmethyl-2,6-dihydro-pyrazolo[4,3- 20 d]pyrimidin-7-one (Compound AA). The results for these experiments are- summarized below and in tabular form (table 1 & 2).

Table 1 012899 52

Ratio Pregabalin: sildenafil Pregabalin (mg/kg) Sildenafil (mg/kg) % reversai of allodynia Total dose Interaction 1:0 30 100 30 0:1 30 50 30 1:1 10 10 100 20 Syn^gy 10:1 10 1 100 11 Synergy

Table 2.

Ratio Gabapentin:Compound AA Gabapentin (mg/kg) Sildenafil (mg/kg) % reversai of allodynia Total dose Interaction 1:0 100 100 100 0:1 30 50 30 10:1 10 | 1 100 11 Synergy

Effect of combinations of pregabalin and sildenafil on CCI-induced statîc 5 allodynia.

Pregabalin and sildenafil had peak anuallodynic actions at 2h postadministration in the CCI-induced static model. Pregabalin and sildenafil wereadministered at fixed dose ratios of 1:1 and 10:1. These fixed dose ratiosdemonstrated synergy with static allodynia completely blocked by a total dose of 10 20mg/kg and 1 lmg/kg respectively. The 1:1 combination represents a 3-fold lowerdose of pregabalin and 3-fold lower dose of sildenafil when administerfed alone whilstthe 1:1 ratio represents a 3-fold lower dose of pregabalin and 30-fold lower dose ofsildenafil when administered alone. 53

Effect of combinations of Gabapentin and Compound AA on CCI-induced static allodynia

Gabapentin and Compound AA had peak antiallodynic actions at 2h postadministration in the CCI-induced static model. Gabapentin and Compound AA were 5 administered at fixed dose ratios of 10:1. This fixed dose ratio demonstrated synergy with static allodynia completely blocked by a total dose of llmg/kg respectively. Thethe 1:1 ratio represents a 10-fold lower dose of gabapentin and 30-fold lower dose ofCompound AA when administered alone. P Suitable PDEV inhibitors of the présent invention may be prepared as described in the aforementioned patent literature references or are obvious to thoseskilled in the art on the basis of these documents.

Suitable alpha-2-delta ligand compounds of the présent invention may be 15 prepared as described herein below or in the aforementioned patent literature references or are obvious to those skilled in the art on the basis of these documents.

Chemistrv Examples 20 Example 1. (3S,5R)-3-Amino-5-methyl-octanoic acid hydrochloride(R)-

. 2,6-Dimethyl-non-2-ene. To (S)-citronellyl bromide (50 g, 0.228 mol) in THF (800 mL) at 0°C was added LiCl (4.3 g) followed by CuCl2 (6.8 g). After 30 minutes méthylmagnésium chloride (152 mL of a 3 M solution in THF, Aldrich) was added and the solution warmed to room température. After 10 hours the solution was cooled 25 to 0°C and a saturated aqueous solution of ammonium chloride carefully added. The1 * résultant two layers were separated and the aqueous phase extracted with ether. Thecombined organic phases were dried (MgSO4) and concentrated to give (R)-2,6-dimethyI-non-2-ene. 32.6 g; 93%. Used without further purification. ’H'NMR (400MHz; CDC13) δ 5.1 (m, 1H), 1.95 (m, 2H), 1.62 (s, 3H), 1.6 (s, 3H), 1.3 (m, 4H), 1.2 30 (m, 2H), 0.8 (s, 6H);13C NMR (100 MHz; CDCI3) 0131.13,125.28, 39.50,37.35, 32.35, 25.92, 25.77, 20.31, 19.74, 17.81, 14.60. 012899 “Γ (R)-4-Methyl-heptanoic acid. Το (R)-2,6-dimethyl-non-2-ene (20 g,0.13 mol) in acetone (433 mL) was added a solution of C1O3 (39 g, 0.39 mol) inH2SO4 (33 mL)/H2O (146 mL) over 50 minutes. After 6 hours a further amount of 5 C1O3 (26 g, 0.26 mol) in H2SO4 (22 mL)/H20 (100 mL) was added. After 12 hoursthe solution was diluted with brine and the solution extracted with ether. Thecombined organic phases were dried (MgSOq) and concentrated. Flashchromatography (gradient of 6:1 to 2:1 hexane/EtOAc) gave (R)-4-methyl-heptanoicacid as an oil. 12.1 g; 65%. MS, m/z (relative intensity): 143 [M-H, 100%]. 10 (4R,5S)-4-Methyl-3-((R)-4-methyl-heptanoyI)-5-phenyl-oxazolidin-2-one.To (R)-4-methyl-heptanoic acid (19 g, 0.132 mol) and triethylamine (49.9 g,0.494 mol) in THF (500 mL) at 0°C was added trimethylacetylchloride (20 g,0.17 mol). After 1 hour LiCl (7.1 g, 0.17 mol) was added followed by (4R,5S)-(+)-4- 15 methyl-5-phenyl-2-oxazolidinone) 3 (30 g, 0.17 mol). The mixture was warmed toroom température and after 16 hours the filtrate was removed by filtration and thesolution concentrated under reduced pressure. Flash chromatography (7:1hexane/EtOAc) gave (4R,5S)-4-methyl-3-((R)-4-methyl-heptanoyl)-5-phenyl-oxazolidin-2-one as an oil. 31.5 g; 79%. [a]p = +5.5 (c 1 in CHCI3). MS, m/z 20 (relative intensity): 304 [M+H, 100%].

(3S,5R)-5-Methyl-3-((4R,5S)-4-methyI-2-oxo-5-phenyl-oxazolidme-3-carbonyB-ortanoic acid tert-butyl ester. To (4R,5S)-4-methyl-3-((R)-4-methyl-heptanoyI)-5-phenyl-oxazolidin-2-one (12.1 g, 0.04 mol) in THF (200 ml) at -50°C 25 was added sodium bis(trimethylsilyl)amide (48 mL of a 1 M solution in THF). After30 min t-butylbromoaceate (15.6 g, 0.08 mol) was added. The solution was stirred for4 hours at -50°C and then warmed to room température. After 16 hours a saturatedaqueous solution of ammonium chloride was added and the two layers separated. Theaqueous phase was extracted with ether and thé combined organic phases dried 30 (MgSC>4) and concentrated. Flash chromatography (9:1 hexane/EtOAc) gave (3S,5R)- 5-methyl-3-((4R,5S)-4-methyl-2-oxo-5-phenyl-oxazolidine-3-carbonyl)-octanoic acid 55 012899 tert-butyl ester as a white solid 12 g; 72%. [<x]g> = +30.2 (c 1 in CHCI3). NMR(100 MHz; CDCI3) δ 176.47, 171.24, 152.72, 133.63, 128.87, 125.86, 80.85, 78.88,55.34, 39.98, 38.77, 38.15, 37.58, 30.60, 28.23, 20.38, 20.13,14.50,14.28. 5 (S)-2-((R)-2-Methyl-pentyl)-succinic acid 4-tert-butyl ester. To (3S,5R)-5- methyl-3-((4R,5S)-4-methyl-2-oxo-5-phenyI-oxazolidine-3-carbonyl)-octanoic acidtert-butyl ester (10.8 g, 0.025 mol) in H2O (73 triL) and THF (244 mL) at 0°C wasadded a premixed solution of LiOH (51.2 mL of a 0.8 M solution) and H2O2a. (14.6 mL of a 30% solution). After 4 hours a further 12.8 mL LiOH (0.8 M solution) 10 and 3.65 mL of H2O2 (30% solution) was added. After 30 minutes sodium bisulfite '(7 g), sodium sulfite (13 g), and water (60 mL) was added followed by hexane(100 mL) and ether (100 mL). The two layers were separated and the aqueous layerextracted with ether. The combined organic phases were concentrated to an oil thatwas dissolved in heptane (300 mL). The résultant solid was filtered off and the filtrate 15 dried (MgSOzp and concentrated to afford (S)-2-((R)-2-methyl-pentyl)-succinic acid4-tert-butyl ester (6 g, 93%) which was used immediately without further purification.MS, m/z (relative intensity): 257 [M+H, 100%]. (3S, 5R)-3-Benzyoxycarbonylamino-5-methyl-octanoic acid, ferf-butyl, 20 ester. A solution of (S)-2-((R)-2-methyl-pentyl)-succinic acid 4-tert-butyl ester (6.0g, 23.22 mmol) and triethylamine (3.64 mL, 26.19 mmol) in toluene (200 mL) wastreated with diphenylphosphoryl azide (5.0 mL, 23.22 mL) and stiired at roomtempérature for 0.5 hours. After the reaction mixture was then heated at reflux for 3hand cooled briefly, benzyl alcohol was added (7.2 mL, 69.7 mmol) and the solution 25 heated for another 3 h. After the reaction mixture was allowed to cool, it was dilutedwith ethyl ether (200 mL) and the combined organic layer was washed successivelywith saturated NaHCCL and brine and dried (NajSCL). The concentrated organiccomponent was purified by chromatography (MPLC) eluting with 8:1 hexanes: ethylacetate to provide (3S, 5R)-3-benzyoxycarbonylamino-5-methyl-octanoic acid, tert- 30 butyl ester (6.4 g, 75.8%). MS: M+l: 364.2, 308.2. 56 (3S, 5R)-3-Amino-5-methyl-octanoic acid, ferf-butyl ester. A solution of(3S, 5R)-3-benzyoxycarbonylamino-5-methyl-octanoic acid, ierf-butyl ester (2.14g,5.88 mmol) in THF (50 mL) was treated with Pd/C (0.2 g) and H2 at 50 psi for 2hours. The reaction mixture was then filtered and concentrated to an oil in vacuo togive (3S, 5R)-3-amino-5-methyl-octanoic acid, ierf-butyl ester in quantitative yield.MS: M+l: 230.2,174.1. (3S, 5R)-3~Amino-5-methyl-octanoic acid hydrochloride. A slurry of (3S,5R)-amino-5-methyl-octanoic acid, terf-butyl ester (2.59g, 11.3 mmol) in 6N HCl(100 mL) was heated under reflux 18 hours, cooled, and filtered over Celite. Thefîltrate was concentrated in vacuo to 25 mL and the resulting crystals were collectedand dried to provide (3S, 5R)-3-amino-5-methyl-octanoic acid hydrochloride, mp142.5-142.7°C (1.2g, 50.56%). A second crop (0.91g) was obtained from the fîltrate.Anal. Calc’d for C9H19NOZHC1: C: 51.55, H: 9.61, N: 6.68, Cl: 16.91. Found: C:51.69, H: 9.72, N: 6.56, Cl: 16.63. (3S, 5R)-3-Amino-5-methyl-octanoic acid hydrochloride acid sait. 5.3 g of2S-(2R-methyl-pentyl)-succinic acid-4-tert-butyl ester contained in 30 mLmethyltertbutyl ether is reacted at room température with 3.5 mL triethylaminefollowed by 6.4 g of diphenylphosphoryl azide. After allowing the reaction toexotherm to 45°C and stining for at least 4 hours, the reaction mixture is allowed tocool to room température and stand while the phases separated. The lower layer isdiscarded and the upper layer is washed with water, followed by dilute aqueous HCl.The upper layer is then combined with 10 mL of 6 N aqueous HCl, and stirred at 45-65°C. The reaction mixture is concentrated by vacuum distillation to about 10 -14mL and allowed to crystallize while cooling to about 5°C. After collecting theproduct by filtration, the product is washed with toluene and reslurried in toluene.

The product is dried by heating under vacuum resulting in 2.9 g (67%) of whitecrystalline product. The product may be recrystallized from aqueous HCl. mp137°C, HNMR (400 MHz, D6 DMSO) δ 0.84 - 0.88 (overlapping d and t, 6H), 1.03 -1.13 (m, 1H), 1.16 -1.37 (m,4H), 1.57 - 1.68 (m, 2H), 2.55 (dd, 1H, J = 7,17 Hz),2.67 (dd, 1H, J = 6,17 Hz), 3.40 (m, 1H), 8.1 ( br s, 3H), 12.8 (br s, 1H).

Û 12899 57

Example 2. (3S, 5R)-Amino-5-methyl-heptanoic acidMethanesulfonic acid (S)-3,7-dimethyI-oct-6-enyl ester. To S-(-)-citronellol (42.8 g, 0.274 mol) and triethylamine (91 mL, 0.657 mol) in CH2CI2 (800 mL) at 0°C5 was added methanesulphonyl chloride (26 mL, 0.329 mol) in CH2CI2 (200 mL).

After 2 hours at 0°C the solution was washed with IN HCl then brine. The organicphase was dried (MgSOzj.) and concentrated to afford the titled compound an oil(60.5 g, 94%) which was used without further purification. MS, m/z (relativeintensity): 139 [100%J, 143 [100%]. '10 (R)-2,6-Dimethyl-oct-2-ene. To methanesulfonic acid (S)-3,7-dimethyl-oct- 6-enyl ester (60 g, 0.256 mol) in THF (1L) at 0°C was added lithium aluminumhydride (3.8 g, 0.128 mol). After 7 hours, a further 3.8 g of lithium aluminum hydridewas added and the solution warmed to room température. After 18 hours, a further 15 3.8 g of lithium aluminum hydride was added. After a further 21 hours, the reaction was carefully quenched with IN citric acid and the· solution diluted further with brine.The résultant two phases were separated and the organic phase was dried (MgSO^.)

20 and concentrated to afford the titled compound as an oil which was used withoutfurther purification. MS, m/z (relative intensity): 139 [M+H, 100%]. (R)-4-Methyl-hexanoic acid. A procedure similar to the synthesis of (R)~4-methyl-heptanoic acid was utilized giving the acid as an oil (9.3 g, 56%). IR (film)2963, 2931, 2877, 2675, 1107, 1461, 1414 cm'1; MS, m/z (relative intensity): 129 [Μ-Η, 100%]. 25 (4R,5S)-4-Methyl-3-((R)-4-methyl-hexanoyl)-5-phenyl-oxazolidin-2-one. A procedure similar to the synthesis of (4R,5S)-4-methyl-3-((R)-4-methyl-heptanoyl)- 5-phenyl-oxazolidin-2-one was utilized giving the titled compound as an oil (35.7 g,95%). MS, m/z (relative intensity): 290 [M+H, 100. 30 (3S,5R)-5-Methyl-3-[l-((4R,5S)-4-methyl-2-oxo-5-phenyl-oxazolidin-3-yI)- methanoylj-heptanoic acid tert-butyl ester. A procedure similar to the préparation 012899 58 of (3S,5R)-5-methyl-3-((4R,5S)-4-methyl-2-oxo-5-phenyl-oxazolidine-3-carbonyl)-octanoic acid tert-butyl ester was followed giving the titled compound as an oil(7.48 g; 31%). MS, m/z (relative intensity): 178 [100%], 169 [100%]; [a]D = + 21.6 (c1 in CHC13). 5 (S)»2-((R)-2-Methyl-butyI)-succinic acid 4-tert-butyl ester. (3S,5R)-5-Methyl-3-[l-((4R,5S)-4-methyl-2-oxo-5-phenyl-oxazolidin-3-yl)-methanoyJ]-heptanoic acid tert-butyl ester (7.26 g, 0.018 mol) in H2O (53 mL) and THF (176 mL)at 0°C was added a premixed solution of LiOH (37 mL of a 0.8 M solution) and 10 H2O2 (10.57 mL of a 30% solution) and the solution warmed to room température.After 2 hours sodium bisulfite (7 g), sodium sulfite (13 g), and water (60 mL) wasadded and the two layers were separated and the aqueous layer extracted with ether.The combined organic phases were concentrated to an oil that was dissolved inheptane (200 mL). The résultant solid was filtered off and the filtrate dried (MgSOzj.) 15 and concentrated to afford the titled compound as an oil (4.4 g) that was used withoutfurther purification. MS, in/z (relative intensity): 243 [100%]. (3S, 5R)-3-Benzyoxycarbonylamino-5-methyl-heptanoic acid, ZcrZ-butylester-This compound was prepared as described above starting with (S)-2-((R)-2- 20 methyl-butyl) succinic acid, 4-tert-butyl ester to give (3S, 5R)-3- benzyoxycarbonylamino-5-methyl-heptanoic acid, tert-butyl ester as an oil (73.3%yield). ’H NMR (400 MHz; CDC13) δ 0.84(t, 3H, J = 7.33 Hz), 0.89(d, 3H, J = 6.60Hz), 1.12-1.38 (m, 4H), 1.41 (s, 9H), 1.43-1.59 (m, 2H), 2.42 (m, 2H), 4.05 (m, 1H),5.07 (t, 2H J =12.95 Hz), and 7.28-7.34 (m, 5H). 25 (3S, 5R)-Amino-5-methyl-heptanoic acid, tert-butyl ester- This compoundwas prepared as described above starting with (3S, 5R)-3-benzyoxycarbonylamino-5-methyl-heptanoic acid, tert-butyl ester instead of (3S, 5R)-3-benzyoxycarbonylamino- 5-methyl-octanoic acid, tezl-butyl ester to give the titled compound. NMR (400 30 MHz; CDCL) δ 0.84 (overlapping t and d, 6H), 1.08-1.16(m, 2H), 1.27-1.30(m, 2H), 012899 59 1.42(s, 9H), 1.62 (br s, 2H), 2.15 (dd, 1H, J= 8.54 and 15.62 Hz), 2.29(dd, 1H, J = 4.15 and 15.37 Hz), and 3.20(br s, 2H). (3S, 5R)-Amino-5-methyl-heptanoic acid hydrochloride-A slurry of (3S, 5 5R)-amino-5-methyl-heptanoic acid, iert-butyl ester (1.44g, 6.69 mmol) in 3N HClwas heated at reflux for 3 hours, filtered hot over Celite, and concentrated to dryness.Trituration of the resulting solid in ethyl ether provided (3S, 5R)-3-amino-5-methyl-heptanoic acid hydrochloride, (0.95g, 85%) mp 126.3-128.3°C. Anal. Calc’d forC8H17N02-HC1-0.1H20: C: 48.65, H: 9.29, N: 7.09, Cl: 17.95. Found: C: 48.61, H: ;0 9.10, N: 7.27, Cl: 17.87MS: M+l: 160.2

Example 3. (3S, 5R)-3-Amino-5-methyi-nonanoic acid(R)-4-Methyl-octanoic acid. Lithium chloride (0.39 g, 9.12 mmol) and copper (I) chloride (0.61 g, 4.56 mmol) were combined in 45 ml THF at ambient 15 température and stirred 15 minutes, then cooled to 0°C at which time ethylmagnesiumbromide (1 M solution in THF, 45 mL, 45 mmol) was added. (S)-citronellyl bromide(5.0 g, 22.8 mmol) was added dropwise and the solution was allowed to warm slowlyto ambient température with stixring ovemight. The reaction was quenched bycautious addition of sat. NH4CI (aq), and stirred with E^O and sat. NH4CI (aq) for 20 30 minutes. The phases were separated and the organic phase dried (MgSC>4) and ) concentrated. The crude (R)-2,6-dimethyl-dec-2-ene was used without purification.

To a solution of (R)-2,6-dimethyl-dec-2-ene (3.8 g, 22.8 mmol) in 50 mL acetone at0°C was added Jones’ reagent (2.7 M in H2SO4 (aq), 40 mL, 108 mmol) and thesolution was allowed to warm slowly to ambient température with stirring ovemight. 25 The mixture was partitioned between F.t20 and H2O, the phases were separated, andthe organic phase washed with brine, dried (MgStXj), and concentrated. The residuewas purified by flash chromatography (8:1 hexanes:EtOAc) to afford-2.14 g (59%) ofthe titled compound as a colorless oil: LRMS: nz/z 156.9 (M+). Jones’ reagent wasprepared as a 2.7M solution by combining 26.7g CrÛ3, 23 mL H2SO4, and diluting to 30 100 mL with H2O. 60 (4R, 5S)-4-Methyl-3-((R)-4-methyl-octanoyI)-5-phenyl-oxazolidin-2-one.Το (R)-4-methyl-octanoic acid (2.14 g, 13.5 mmol) in 25 mL CH2CI2 at 0°C wasadded 3 drops DMF, followed by oxalyl chloride (1.42 mL, 16.2 mmol) resulting in 5 vigorous gas évolution. The solution was wanmed directly to ambient température,stiired 30 minutes, and concentrated. Meanwhile, to a solution of the oxazolidinone(2.64 g, 14.9 mmol) in 40 mL THF at -78°C was added n-butyllithium (1.6 M soin inhexanes, 9.3 mL, 14.9 mmol) dropwise. The mixture was stiired for 10 minutes atwhich time the acid chloride in 10 mL THF was added dropwise. The reaction was 10 stirred 30 minutes at -78°C, then warmed directly to ambient température andquenched with sat. NH4CI. The mixture was partitioned between Et2Û and sat.NH4CI (aq), the phases were separated, and the organic phase dried (MgSOq), andconcentrated to fumish 3.2 g of the titled compound as a colorless oil. LRMS: m/z 318.2 (M+). 15 (3S,5R)-5-Methyl-3-((4R,5S)-4-methyl-2-oxo-5-phenyI-oxazoIidine-3-carbonyl)-nonanoic acid tert-butyl ester. To a solution of diisopropylamine(1.8 mL, 12.6 mmol) in 30 mL THF at -78°C was added n-butyllithium (1.6 M soin inhexanes, 7.6 mL, 12.1 mmol), and the mixture stirred 10 minutes at which time (4R, 20 5S)-4-Methyl-3-((R)-4-methyl-octanoyl)-5-phenyl-oxazolidin-2-one (3.2 g, 10.1 mmol) in 10 mL THF was added dropwise. The solution was stirred for30 minutes, Z-butyl bromoacetate (1.8 mL, 12.1 mmol) was added quickly dropwise at-50°C, and the mixture was allowed to warm slowly to 10°C over 3 hours. Themixture was partitioned between Et2Ü and sat. NH4CI (aq), the phases were 25 separated, and the organic phase dried (MgSOq), and concentrated. The residue waspurified by flash chromatography (16:1 to 8:1 hexanes:EtOAc) to provide 2.65 g(61%) of the titled compound as a colorless crystalline solid, mp = 84-86°C. [δ]ρ23+17.1 (c = 1.00, CHCI3). 30 (S)-2-((R)-2-MethyI-hexyI)-succinic acid 4-tert-butyl ester. To a solution of (3S,5R)-5-Methyl-3-((4R,5S)-4-methyl-2-oxo-5-phenyl-oxazolidine-3-carbonyl)- 61 nonanoic acid tert-butyl ester (2.65 g, 6.14 mmol) in 20 mL THF at 0°C was added aprecooled (0°C) solution of LiOH monohydrate (1.0 g, 23.8 mmol) and hydrogenperoxide (30 wt% aqueous soin, 5.0 mL) in 10 mL HgO. The mixture was stirredvigorously for 90 minutes, then warmed to ambient température and stirred 5 90 minutes. The reaction was quenched at 0°C by addition of 100 mL 10% NaHSOg (aq), then extracted with Et20. The phases were separated, and the organic phasewashed with brine, dried (MgSOzj), and concentrated. The titled compound was usedwithout purification. 10 (3S, 5R)-3-Benzyoxycarbonylamino-5-methylnonanoic acid, tert-butyl

ester-This compound was prepared similarly as described above starting with (S)-2-((R)-2-methylhexyl) succinic acid, 4-/ert-butyl ester instead of (S)-2-((R)-2-methylpentyl) succinic acid, 4-tert-butyl ester to provide the titled compound as an oil(71.6% yield). 1HNMR (400 MHz; CDC13) δ 0.81(t, 3H, J= 4.40 Hz), 0.85(d, 3H, J 15 = 6.55 Hz), 1.06-1.20(m, 7H), 1.36(s, 9H), 1.38-1.5O(m, 2H), 2.36(m, 2H), 3.99(m, 1H), 5.02(m+s, 3H), and 7.2S-7.28(m, 5H). (3S, 5R)-3-Àmino-5-methyl-nonanoic acid, tert-butyl ester- This compoundwas prepared as described above starting with (3S, 5R)-benzyoxycarbonylamino-5- 20 methyl-nonanoic acid, zerf-butyl ester instead of (3S, 5R)-3-benzyoxycarbonylamino- 5-methyl-octanoic acid, tert-butyl ester. Yield = 97%. ’HNMR (400 MHz; CDC13) δ0.82(overlapping d and t, 6H), 1.02-1.08(m, 1H), 1.09-1.36(m, 6H), 1.39(s, 9H),1.47(br s, 1H), 1.80(s, 2H), 2.13(dd, 1H, J = 8.54 and 15.61 Hz), and 2.27(dd, 1H, J =4.15 and 15.38 Hz). 25

(3S, 5R)-3-Amino-5-methyl-nonanoic acid hydrochloride-A mixture of (3S,5R)-3-amino-5-methyl-nonanoic acid, tert-butyl ester (1.50g, 6.16 mmol) in 3N HCl(100 mL) was heated at reflux for 3hours, filtered hot over Celite, and concentrated to30mL in vacuo. The resulting crystals were collected, washed with additional 3N 30 HCl, and dried to provide the title compound, mp 142.5-143.3°C. Additional cropswere obtained from the filtrate to provide 1.03g (70.4%). Anal. Calc’d for 012899 62 C10H2iNO2 HC1: C: 53.68, H: 9.91, N: 6.26, Cl: 15.85. Found: C: 53.89, H: 10.11, N:6.13. MS: M+l:188.1.

Example 4, (2R, 4R)-2-Aminoniethyl-4-methyl-heptanoic acid5 5R-Methyl-3R-(4S-methyl-2-oxo-5R-phenyloxazolidine-3- carbonyl)octanoic acid. A solution of (3R,5R)-5-Methyl-3-((4S,5R)-4-methyl-2-oxo-5-phenyl-oxazolidine-3-carbonyl)-octanoic acid tert-butyl ester(3.9 g, 9.34mmol) in dichloromethane (150 mL) was treated with trifluoroacetic acid (7.21 mL,93.4 mL) and stirred 18 hours at ambient température. After the solvents and reagent 10 were removed in vacuo, the resulting residue was tritrurated in 100 mL hexanes toprovide 3.38g of the title compound (100%) mp 142-143°C.

[4R-Methyl-2R-(4S-methyl-2-oxo-5R-phenyloxazolidine-3-carbonyl)heptyl]carbamic acid benzyl ester. A solution of 5R-methyl-3R-(4S- 15 methyl-2-oxo-5R-phenyloxazolidine-3-carbonyl)octanoic acid (1.98 g, 5.48 mmol)and triethylamine (0.92mL, 6.57 mmol) was treated with diphenylphosphorylazide(1.2 mL, 5.48 mmol), stirred 30 min at ambient température and then heated at refluxfor 3 hours. After cooling briefly, the reaction mixture was treated with benzylalcohol (2.8 mL, 27.4 mmol) and heated for an additional 3 h at reflux. The reaction 20 mixture was cooled, diluted with ethyl ether (150 mL), washed successively withsat’d NaHCC>3 and brine, dried (MgSCh) and concentrated in vacuo to an oil.Chromatography (MPLC, elution with 4:1 hexanes:ethyl acetate) provided the titlecompound (2.0 g, 78,3%) as an oil. MS M+l = 467.1.

25 2R-(BenzyloxycarbônyIaminomethyI)-4R-methyIheptanoic acid. A solution of 4R-methyl-2R-(4S-methyl-2-oxo-5R-phenyloxazolidine-3-carbonyl)heptyl]carbamic acid benzyl ester (4.12 g, 8.83 mmol) in 3:1 THF: water(100 mL) was cooled to 0°C and treated with a mixture of 0.8 N LiOH (17.5 mL, 14mmol) and 30% H2O2 (4.94 mL, 44 mmol). After the reaction mixture was stirred in 30 the cold 3hours, it was quenched with a slurry of NaHSCb (2.37g) and Na^SOs (4.53g)in water (30 mL) and stirred 1 hour. The reaction mixture was diluted with ethyl ether(200 mL), partitioned, and the organic layer washed with brine and dried (MgSO4). 63

The concentrated organic extract was chromatographed (MPLC) eluting with ethylacetate to give 1.25g of 2R-(benzyloxycarbonylaminomethyl)-4R-methylheptanoicacid (46%). MS M+l = 308.1. 5 (2R,4R)-2-Àmino-4-methyI-hep(anoic acid hydrochloride. A mixture of 2R-(benzyloxycarbonylaminomethyl)-4R-methyl-heptanoic acid (1.25g, 4.07 mmol)and Pd/C (20%, 0.11 g) in methanol (50 mL) was hydrogenated at 50 psi for 18 hours.After the catalyst was removed by filtration, the solvent was removed in vacuo andthe resulting solid triturated in ether to provide (2S, 4R)-2-amino-4-methyl-heptanoic )0 acid hydrochloride (0.28g, 40%) mp 226.3-228.0°C. MS M+l = 174.0. Anal. Calc’dfor C9Hi9NO2-0.1 H2O C: 61.75 H: 11.06 N: 8.00. Found C: 61.85 H: 10.83 N: 8.01.

Example 5.2-Aminomethyl-4,4-dimethyl-heptanoic acid hydrochloride. 2-Cyano-4,4-dimethyl-hepta-2,6-dienoic acid ethyl ester. A solution of 2,2- 15 dimethyl-pent-4-enal (5.0g, 44 mmol), cyano-acetic acid ethyl ester (5.12 mL, 48mmol), piperidine (1.3 mL, 14 mmol) and acetic acid (4.52 mL, 80 mmol) in 170 mLof toluene was heated under reflux for 18 hours in a flask equipped with aDean-Starkseparator. Several mL of water was collected in the trap. The reaction was cooled andwashed with IN HCl, NaHCO3 and brine, successively. The organic layers were dried

20 over Na2SÛ4 and concentrated to an oil. This oïl was chromatographed eluting with20% of EtOAc in hexane to give a combination of two lots total 8.3g (91%). ’H NMR ' (400 MHz; CDC13) 1.28 (s, 6H), 1.32 (t, 3H, J = 7 Hz),. 2.26 (d, 2H, J = 7.6 Hz), 4.27 (q, 2H, J = 7.2 Hz), 5.08 (d, 1H, J = 12 Hz), 5.10 (d, 1H, J = 4 Hz), 5.72 (m, 1H). 25 2-Àminomethyl-4,4-dimethyi-heptanoic acid hydrochloride. 2-Cyano-4,4- dimethyl-hepta-2,6-dienoic acid ethyl ester (5.8 Sg, 28 mmol) was dissolved in themixture of 91 mL of éthanol and 6 mL of HCl and treated with 0.4g of PtO2. Thereaction was camed out under 100 psi of hydrogen pressure at room-température for15 hours. The catalyst was filtered and filtrate was concentrated to give 3.8g of the 30 desired product 2-aminomethyl-4,4-dimethyl-heptanoic acid ethyl ester as an oil. MS(APCI): 216.2 (M+l)+. This oil was refluxed in 75 mL of 6N HCl for 18 hours. Whilethe reaction was cooled, a precipitate formed. The solid was filtered, washed with 012899 64 additional HCl solution and triturated with ether to give the clean title compound. MS(APCI): 188.1 (M+l)+. 186.1 (M-l)+. ’H NMR (400 MHz; CD3OD): 0.91 (9H, m), 1.30 (5H, m), 1.81 (dd, 1H, J = 7.2 Hz, 14.4 Hz), 2.72 (1H, m), 3.04 (2H, m); Anal.

Calc’d for C10H21NO2-HCl: C: 53.68, H: 9.91, N: 6.26, Cl: 15.85; Found: C: 53.83, H: 5 10.15, N: 6.22, Cl: 15.40. MP: 229.5-231.0°C.

Example 6. (£)-3-Amino-5,5-dimethyl-octan.oic acid. 3-(4,4-Dimethyl-heptanoyl)-(Æ)-4-methyl-(5')-5-phenyl-oxazolidin-2-one: A solution of 4,4-dimethyl-heptanoic acid (1.58g, lOmmol) and triethylamine (4.6 10 mL) in 50 mL THF was cooled to 0°C and treated with 2,2-dimethyI-propionyl ( " chloride (1.36 mL). After one hour, 4R-methyl-5S-phenyl-oxazolidin-2-one (1.95g, 1 Immol) and lithium chloride (0.47g, 11 mmol)was added and the mixture was stirred · · ' for 18 hours. The precipitate was filtered and washed thoroughly with additional THF.

The filtrate was concentrated in vacuo to give an oily solid. This solid was dissolved 15 in 200 mL Et2O, washed successively with saturated NaHCO3, 0.5N HCl andsaturated NaCl, dried (MgSO4) and concentrated in vacuo to give the title compoundas an oil (3.0g, 95%). ’HNMR (400 MHz; CDC13): 0.73-0.84 (m, 12H), 1.10-1.22 (m, 4H), 1.46-1.54 (m, 2H), 2.75-2.87 (m, 2H), 4.70 (m, 1H, J= 7 Hz), 5.59 (d, 1H,7 = 7Hz), 7.22-7.37 (m, 5H). 20 5,5-Dimethyl-(£)-3-((f?)-4-methyl-2-oxo-(£)-5-phenyl-oxazolidine-3-carbonyl)-octanoic acid tert-butyl ester: According to example 1, 5.07g (16 mmol)of 3-(4,4-dimethyl-heptanoyl)-4-methyl-5-phenyî-oxazolidin-2-one, 18 mL (IN, 18mmol) of NaHMDS solution and 4.72 mL (32 mmol) of bromo-acetic acid tert-butyl 25 ester gave 3.40g (49.3%) of the title compound as a crystalline solid. m.p.: 83-85°C. (£)-2-(2,2-DimethyI-pentyl)-succinic acid 4-tert-butyI ester: According toexample 1, 3.4g (7.9 mmol) of 5,5-dimethyl-3-(4-methyl-2-oxo-5-phenyl-oxazolidine-3-carbonyl)-octanoic acid tert-butyl ester, 16 mL (12.8 mmol) of 0.8N LiOH and 4.5 f

30 mL of 30% H2O2 gave 2.42g (>100%) of the title compound as an oil. 1HNMR (400 I MHz; CDC13): 0.77-0.82 (m, 9H), 1.14-1.29 (m, 5H), 1.42 (s, 9H), 1.77 (dd, 1H, 7= 8 {

I ! i 65

Hz, 16 Hz), 2.36 (dd, 1H, J = 6 Hz, 16 Hz), 2.59 (dd, 1H, J = 8 Hz, 16 Hz), 2.75-2.85 (m, 1H). (S)-3-BenzyIoxycarbonyIamino-5,5-dimethyl-octanoic acid tert-butyl 5 ester: According to example 1, 2.14g (7.9 mmol)of 2-(2,2-dimethyl-pentyl)-succinicacid 4-tert-butyl ester, 1.7 mL of DPPA, 1.1 mL of EtsN and 2.44 mL of BnOHprovided 1.63g (54.8% in two steps) of the title compound as an oil. ’HNMR (400MHz; CDCI3): 0.78-0.89 (m, 9H), 1.10-1.30 (m, 5H), 1.36 (s, 9H), 2.39 (t, 2H, 7 = 5Hz), 4.95-4.05 (m, 1H), 5.00 (s, 2H), 5.09 (d, 1H, 7=9.6 Hz), 7.22-7.30 (m, 5H). (S)-3-Amino-5,5-dimethyl-octanoic acid tert-butyl ester: According toexample 1, 1.63g of 3-benzyloxycarbonylamino-5,5-dimethyl-octanoic acid tert-butylester and 0.2g of 20% Pd/C fumished the title compound. MS, m/z, 244.2 (M+l)+. 15 (S)-3-Arnino-5,5-dimethyl-oc.tanoic acid hydrochloride: According to example 1, 3-amino-5,5-dimethyl-octanoic acid tert-butyl ester was treated with 3NHCl to provide 286mg of the title compound as a solid. MS (APCI), m/z: 188.1(M+l)+. 186.1 (M-l)+. Anal. Calc’d for ^0Η2ΙΝΟ2Ήα·0.12Η2Ο: C: 53.17, H: 9.92,N: 6.20, Cl: 15.69; Found: C: 53.19, H: 10.00, N: 6.08, Cl: 15.25. a = +20° (MeOH). 20 MP: 194.2-195.2°C.

Example 7. 2-AminomethyI-3-(l-methyl-cydopropyl)-propionic acid.2-Cyano-3-(l-methyl-cyelopropyl)-acryIic acid ethyl ester. To 1- methylcyclopropane-methanol (Aldrich, 1.13mL, 11.6mmol) in 50mL CH2CI2 was 25 added neutral alumina (2.5g) and then PCC (2.5g, 11.6 mmol), and the mixture stirred3h at ambient température. The mixture was filtered through a 1cm plug of silica gelunder vacuum, and rinsed with Et2O. The filtrate was concentrated to ca. 5mL totalvolume. To the residue was added THF (lOmL), ethyl cyanoacetdte (1.2mL, 11.3mmol), piperidine (5 drops), and finally acetic acid (5 drops). The whole was stirred 30 at ambient température ovemight, then partitioned between Et2O and sat. aq.NaHCCh. The phases were separated and the organic phase washed with brine, dried(MgSO4), and concentrated. Flash chromatography of the residue (10—?15% 66 012899

EtOAc/hexanes) provided 0.53g (25%) of the ester as a colorless oil that crystallizedon standing. Anal. Calcd for C10Hi3NO2: C, 67.02; H, 7.31; N, 7.82. Found: C, 66.86; H, 7.47; N, 7.70. 2-Aminomethyl-3-(l-methyl-cycIopropyl)-propionic acid ethyl ester. To 2-cyano-3-(l-methyl-cyclopropyl)-acrylic acid ethyl ester (0.45g, 2.5Immol) in 16mL EtOHTHF (1:1) was added RaNi (0.4g), and the mixture was hydrogenated in aParr shaker at 48 psi for 15.5 h. Pearlman’s catalyst (0.5g) was then added andhydrogénation was continued for an additional 15h. The mixture was filtered andconcentrated. Flash chromatography of the residue 2—>3—>4—>5—>6—>8% . |

MeOH7CH2Cl2 provided 0.25g (54%) of the aminoester as a colorless oil. LRMS:m/z 186.1 (M+l). 2-Aminomethyl-3-(l-niethyl-cyclopropyl)-propionic acid. To a solution of2-aminomethyl-3-(l-methyl-cyclopropyl)-propionic acid ethyl ester (0.25g,1.35mmol) in lOmL methanol at 0°C was added 10% aq. NaOH (lOmL). Themixture was stirred at ambient température ovemight, then concentrated to remove themethanol. The residue was cooled to 0°C and acidified to pH 2 with conc. HCl.

After allowing to warm to ambient température the mixture was loaded ontoDOWEX-50WX8-100 ion exchange resin and eluted with H2O until neutral to litmus.

Elution was continued with 5% aq. NH4OH (lOOmL) and the alkaline fractions "concentrated to provide 0.15g (71%) of the amino acid as a colorless solid. LRMS:m/z 158.0 (M+l).

Example 8. (35,5R)-3-Amino-5-methyl-octanoic acid. (5S)-5-Methyl-octa-2,6-dienoic acid tert-butyl ester. To a solution of (5)-3-methyl-hex-4-enoic acid ethyl ester* (1.0g, 6.4mmol) in 30mL toluene at -78 °C wasadded DIB AH (1.0M in THF, 6.4mL) dropwise over 5 min. The mixture was stirredat -78 °C 45 min at which time 5 drops of methanol were added, resulting in vigorousH évolution. Methanol was added until no more gas évolution was observed (ca. 5mL). At this time the cold bath was removed and ca. 5mL of sat. aq. Na+K+ tartratewas added. When the mixture reached room température, additional sat. aq. Na+K+ 67 012899 tartrate and Et2O were added and stirring was continued until the phases were mostlyclear (ca. lh). The phases were separated, and the organic phase washed with brine,dried (MgSCh), and concentrated to ca. lOmL total volume owing to volatilityconcems. The crude mixture was combined with an additional batch of aldéhyde 5 prepared from lOmmol of the ester by the method described above and the whole usedwithout purification. To a suspension of sodium hydride (60% dispersion in mineraioil) in 25mL THF was added i-butyl-7’,P-dimethylphosphonoacetate (3.0mL,15mmol) dropwise over lh such that the évolution of H2 was under control. After theaddition was complété, the crude aldéhyde in toluene (ca. 20mL total volume) was 10 added quickly dropwise and the mixture stirred at ambient température ovemight.The mixture was partitioned between Et2O and sat. aq. NH4CI, the phases separated,the organic phase washed with brine, dried (MgSCh), and concentrated. Flashchromatography of the residue (0—>3—>5% EtOAc/hexanes) afforded 1.0g (29%, twosteps) of the unsaturated ester as a pale yellow oil: ’lT NMR (CDCI3) δ 6.75 (m, 1H), 15 5.66 (m, 1H), 5.30 (m, 2H), 2.03-2.29 (m, 3H), 1.58 (d, 7= 6.1 Hz, 3H), 1.41 (s, 9H), 0.91 (d, 7= 6.6 Hz, 3H). *(S)-3-methyl-hex-4-enoic acid ethyl ester was prepared from (S)-trans-3-Penten-2-ol [Liang, J.; Hoard, D. W.; Van Khau, V.; Martinelli, M. J.; Moher, E. D.; 20 Moore, R. E.; Tius, M. A. 7. Or g. Chem., 1999, 64, 1459] via Johnson-Claisen| rearrangement with triethylorthoacetate according to the literature protocol [Hill, R. K.; Soman, R.; Sawada, S., 7. Org. Chem., 1972, 37, 3737]. (3R,5S)-3-[Benzyl-(l-phenyl-ethyl)-ammo]-5-methyl-oct-6-enoic acid tert- 25 butyl ester. To a solution of (5)-(-)-A-benzyl-oc-methylbenzylamine (0.60mL,2.85mmol) in 9.0mL THF at -78 °C was added n-butyllithium (1.6M in hexanes, 1.6mL) quickly dropwise resulting in a deep pink color. The mixture was stirred at -78°C for 30 min at which time (5S)-5-Methyl-octa-2,6-dienoic acid tert-butyl ester(0.5g, 2.38mmol) in l.OmL THF was added slowly dropwise, resulting in a pale tan 30 color which darkened over 3h. The mixture was stirred 3h at -78 °C, then quenchedwith sat. aq. NH4CI. The mixture was allowed to warm to rt and stirred ovemight,then partitioned between EtOAc and sat. aq. NH4CI. The phases were concentrated, 68 042899 and the organic phase dried (MgSCL), and concentrated. Flash chromatography of theresidue (3—>5% EtOAc/hexanes) provided 0.52g (52%) of the aminoester as a yellowoil. ]H NMR (CDCI3) δ 7.34 (m, 2H), 7.20 (m, 8H), 5.27 (m, 2H), 3.74 (m, 1H), 3.72(d, J = 15.9 Hz, 1H), 3.41 (d, J = 14.9 Hz, 1H), 3.27 (m, 1H), 2.38 (m, 1H), 1.98 (dd,J = 3.7, 14.2 Hz, 1H), 1.81 (dd, J = 9.3, 14.4 Hz, 1H), 1.54 (d, J = 4.9 Hz, 3H), 1.32(s, 9H), 1.24 (d, J = 7.1 Hz, 3H), 0.99 (m, 2H), 0.74 (d, J = 6.6 Hz, 3H). (35,5jR)-3-Airdno-5-methyI-octanoic acid. To a solution of (3R,55)-3-[Benzyl-(l-phenyl-ethyl)-amino]-5-methyl-oct-6-enoic acid tert-butyl ester (0.92g,2.18nunol) in 50mL MeOH was added 20% Pd/C (0.20g), and the mixture washydrogenated in a Parr shaker at 48 psi for 23 h. The mixture was filtered andconcentrated. To the crude aminoester in lOmL CH2CI2 was added l.OmLtrifluoroacetic acid, and the solution stirred at ambient température ovemight. Themixture was concentrated, and the residue dissolved in the minimum amount of H2O,and loaded onto DOWEX-50WX8-100 ion exchange resin. The column was elutedwith H2O until neutral to litmus, then continued with 5% aq. NH4OH (lOOmL). Thealkaline fractions were concentrated to provide 0.25g (66%, two steps) of the aminoacid as an off-white solid. *H NMR (CD3OD) δ 3.41 (m, 1H), 2.36 (dd, J = 5.1, 16.6Hz, 1H), 2.25 (dd, J = 8.1, 16.6 Hz, 1H), 1.42 (m, 2H), 1.24 (m, 1H), 1.12 (m, 2H), 1.00 (m, 1H), 0.73 (d, J = 6.4 Hz, 3H), 0.68 (t, J = 6.8 Hz, 3H). LRMS: m/z 172.1(M-l). “ |

Example 9. 2-Aminomethyl-8-methyl-nonanoic acid. A procedure similar to that of 2-Aminomethyi-4,4,8-trimethyl-nonanoic acidwas utilized to préparé 2-Aminomethyl-8-methyl-nonanoic acid from 6-methyl-l-heptanol m/z 202.1 (M+). 2-AminomethyI-4,8-dimethyl-nonanoic acid (R)-2,6-dimethyl heptan-l-ol: Magnésium tumings (2.04 g, 84 mmol) and acrystal of iodine were suspended in 5 mL THF for the addition of l-bromo-3-methylbutane (0.3 mL, neat). The mixture was heated to start the Grignard formation. Theremaining l-bromo-3-methyl butane (8.63 mL, 72 mmol) was diluted in THF (60 ml.) 69 012899 and added dropwise. The mixture was stirred at ambient température for 2 hours andcooled to -5 °C. A solution of copper chloride (1.21 g, 9 mmol) and LiCl (0.76 g, 18mmol) in THF (50 τη T) was added dropwise keeping the température below 0 °C.The resulting mixture was stirred for 20 min, and (R)-3-bromo-2-methylpropanol in 5 THF (20 mï.) was added dropwise while keeping the température below 0 °C. Themixture was allowed to slowly reach ambient température ovemight. The reactionmixture was quenched with ammonium hydroxide and water. The mixture wasdiluted with EtOAc and extracted with 3x20 mL EtOAc. The organics were washedwith brine, dried (MgSCU), filtered and concentrated. The residual oil was purified via 10 silica gel chromatography (90/10 Hexane/EtOAc) to give 2.67 g (R)-2,6-dimethylheptan-l-ol. (R)-l-iodo-2,6-dimethyl heptane: To a mixture of supported triphenylphosphine (6.55 g, 19.67 mmol) in CH2CI2 at 0 °C was added iodine (4.99 g, 19.67 15 mmol) and imidazole (1.33 g, 19.67 mmol). The mixture was warmed to ambienttempérature, stirred for 1 h and cooled to 0 °C for the dropwise addition of (R)-2,6-dimethyl heptan-l-ol in CH2CI2 (5 mL). The mixture was allowed to reach ambienttempérature and stirred for 1 h, at which time it was filtered through a pad of celiteand the solids were washed with CH2CI2. The filtrated was concentrated, and the 20 crude product was purified via silica gel chromatography to give (R)-l-iodo-2,6-dimethyl heptane (2.44 g).

(4R)-4,8-dimethyI nonanoic acid t-butyl ester: To diisopropyl amine (0.827mL, 5.9 mmol) in THF (8 mL) at -78 °C was added nBuLi (2.65 mL of a 2.6 M 25 solution in pentane). The solution was stirred for 30 min at -78 °C, followed by theaddition of t-butyl acetate (0.8 mL, 5.9 mmol). The mixture was stirred at -78 °C for2 h, and then (R)-l-iodo-2,6-dimethyl heptane (0.3 g, 1.18 mmol) and HMPA (1.5mL) in THF (1 mL) was added. The reaction was stirred at -78 °C and allowed toslowly reach ambient température ovemight, then heated at 35 °C to drive the reaction 30 to completion. The reaction was quenched by the addition of ammonium chloride(saturated aqueous solution), and the mixture was extracted with EtOAc (2x10 mT.) Ü12899 70

The organics were combined, washed with water, dried (MgSO4), filtêred andconcentrated. Silica gel chromatography (98/2 hexane/EtOAc) provîded 0.25 g of(4R)-4,8-dimethyl nonanoic acid t-butyl ester. (4R)-4,8-dimethyl nonanoic acid: (4R)-4,8-dimethyl nonanoic acid t-butylester in 25 mL CH2C12 at 0 °C was treated with TFA (6 mL). The mixture wasallowed to reach ambient température and stir ovemight. The solvent was removedby rotary évaporation, and the mixture was purified by silica gel chromatography(95/5 hexane/EtOAc) to give 0.962 g (4R)-4,8-dimethyl nonanoic acid, m/z 185 (M-).

3-(4R,8-Dimethyl-nonanoyl)-4(S)-methyl-5(R)-phenyI-oxazolidin-2-one: A procedure similar to (4R,5S)-4-Methyl-3-(R)-4-methyl-heptanoyl)-5-oxazolidin-2-onewas utilized to give 3-(4R,S-Dimethyl-nonanoyl)-4(S)-methyl-5(R)-phenyl-oxazolidin-2-one (1.35 g) m/z 346.5 (M+). .

[4R,8-Dimethyl-2R-(4R-methyl-2-oxo-5R-phenyl-oxazolidine-3-carbonyl)-nonyl]-carbamic acid benzyl ester: To a solution of 3-(4(R),8-Dimethyl-nonanoyl)-4(S)-methyl-5(R)-phenyl-oxazolidin-2-one (1.05 g, 3.04 mmol) in CH2C12 (12 mL)and T1CI4 (3.04 mL of a 1 M solution in CH2C12) was added diisopropyl ethyl amine(0.55 mL, 3.19 mmol)at -20 °C. The resulting darkred solution was stirred at-20 °Cfor 30 min prior to the addition of a solution of N-methoxymethyl benzyl carbamate(0.652 g, 3.34 mmol) in CH2C12 (3.5 mL) and TiCl4 (3.34 mL). The mixture wasstirred at 0 °C for 4 h. The reaction was quenched by the addition of saturatedaqueous ammonium chloride solution. The mixture was extracted with CH2C12 (3x15mL). The organics were combined and washed with 1 N HCl and neutralized withNaOH, followed by washing with brine. The organics were dried (MgSO4), filtered,concentrated and purified by silica gel chromatography (95/5 hexane /EtOAc) to give0.555 g [4R,8-Dimethyl-2R-(4R-methyl-2-oxo-5R-phenyl-oxazolidine-3-carbonyl)-nonyl]-carbamic acid benzyl ester.

2(R)-(BenzyIoxycarbonylamino-methyI)-4(R),8-dimethyl-nonanoic acid: A procedure similar to that of (S)-2-((R)-2-Methyl=pentyl)succinic acid t-butyl ester 012899 71 was utilized to provide 0.198 g 2(R)-(Benzyloxycarbonylamino-methyl)-4(R),8- dimethyl-nonanoic acid. 2-aniinomethyl-4,8-dimethyI nonanoic acid: 2(R)- 5. (Benzyloxycarbonylamino-methyl)-4(R),8-dimethyl-nonanoic acid (0.148 g, 0.566mmol) was treated with hydrogen in the presence of 20% pd/C to give 0.082 g of 2-aminomethyl-4,8-dimethyl nonanoic acid after filtration and purification via silica gel chromatography (85/15 CHCL/MeOH). m/z 216.3 (M+). |0 Example 10. 2-AminomethyI-4,4,8-trimethyl-nonanoic acid. 2.2.6- Trimethyl-heptanoic acid methyl ester: To diisopropyl amine (1.54mL, 11.03 mmol) in THF (22 mL) at -78 °C was added nBuLi (6.89 mL of a 1.6 Msolution in hexane). The solution was stirred for 30 min at -78 °C, followed by theaddition of methyl isobutyrate (0.97 mL, 8.48 mmol). The mixture was stirred at -78 15 °C for 2 h, and then l-iodo-4-methyl pentane (1.8 g, 8.48 mmol) andDMPU (0.55mL, 4.24 mmol) in THF (6 mL) was added. The reaction was stirred at -78 °C andallowed to slowly reach ambient température over 16 h. The reaction was quenchedby the addition of ammonium chloride (saturated aqueous solution), and the mixturewas extracted with EtOAc (2x10 mL). The organics were combined, washed with 20 water, dried (MgSO4), filtered and concentrated. Silica gel chromatography (99/1h hexane/EtOAc) provided 1.57 g of 2,2,6-Trimethyl-heptanoic acid methyl ester.

2.2.6- Trimethyl heptan-l-ol: 2,2,6-Trimethyl-heptanoic acid methyl ester(1.97 g, 10.6 mmol) was taken up in toluene (65 mL) and cooled to -78 °C. DiRAT.H

25 (12.7 mL of a 1 N solution in toluene) was added dropwise. After 45 min, 1.5 mL

DiBALH was added. After 2 h, the reaction was quenched by the addition of 15 mLMeOH at -78 °C. The mixture was warmed to ambient température, and then cooledagain to -78 °C for the addition of 10 mL 1 N HCl. The mixture was extracted withEtOAc (3x15 mL). The combined organics were washed with brine, dried (MgSO4), 30 filtered and concentrated. The residual oil was purified via silica gel chromatography(95/5 Hexane/EtOAc) to give 2,2,6-Trimethyl-heptan-l-ol (0.88 g), in/z 159 (M-t-). 012899 72 2,2,6-Trimethyl-heptanal: Pyridinium chlorochromate (PCC, 4.17 g, 19.4mmol) was combined with neutral alumina (14.6 g) in CH2CI2 and stirred at ambienttempérature for 15 min. The alcohol was diluted in CH2CI2, and the mixture wasstirred at ambient température for 2h. The solution was filtered through a pad of silica, 5 and the solids were washed with CH2CI2. The filtrate was evaporated to givel.05 gm/z 157 (M+).2,2,6-Trimethyl-heptanal which was carried on without furtherpurification. 2-Cyano-4,4,8-trimethyl-non-2-enoic acid benzyl ester: To a mixture of 10 2,2,6-Trimethyl-heptanal (1.05 g, 6.73 mmol), piperidine (0.19 mL, 2.01 mmol) andbenzyl cyanoacetate (1.29 g, 7.4 mmol) in toluene (50 mL) was added glacial aceticacid (0.72 g, 12.1 mmol). The flask was fitted with a Dean-Stark trap, and themixture was heated at reflux for 18. The mixture was cooled, treated with dilute HCl,and the layers were separated. The organics were washed with a saturated sodium 15 bicarbonate solution followed by brine, and dried (MgSCU), filtered and concentrated.The residual oil was purified by silica gel chromatography (98/2 hexane/EtOAc) togive 1.3 g of 2-Cyano-4,4,8-trimethyl-non-2-enoic acid benzyl ester m/z. 314 (M+).

2-aininomethyI-4,4,8-trimethyI-nonanoic acid: 2-Cyano-4,4,8-trimethyl-20 non-2-enoic acid benzyl ester (1.3 g, 4.14 mmol) in THF (50 mL) was treated withhydrogen in the presence of 20% Pd/C to give a mixture of the cyano acid and thecyano metliyl ester. The mixture was purified by silica gel chromatography to give278 mg of 80105x41-1-2. The acid was then treated with hydrogen in the presence ofRaney Ni in MeOH7NH4OH to give 0.16 g of 2-aminomethyl-4,4,8-trimethyl- 25 nonanoic acid, m/z 230.3 (M+).

Example 11. 2-ÀminomethyI-4-ethyl-octanoic acid. A procedure similar to that of 2-Aminomethyl-4,4,8-trimethyl-nonanoic acid was utilized to préparé 2-Aminomethyl-4-ethyl-octanoic acid from 2-ethylhexanal. 30 m/z 202.1 (M+).

Example 12. 2-Aminomethyl-4-ethyI-8-methyl-nonanoic acid. 012899 73 \

A A procedure similar to that of 2-Aminomethyl-4,4,8-trimethyl-nonanoic acidwas utilized to préparé 2-Aminomethyl-8-methyl-nonanoic acid from 2,6-di-t-butyl-4-methylphenyl cyclopropylcarboxylate. rn/z 230.2 (M+). 5 Example 13. 3-Ammo-2-[l-(4-methyl-pentyl)-cyclopropylmethyl]- propionic acid. A procedure similar to that of 2-Aminomethyl-4,4,8-trimethyl-nonanoic acidwas utilized to préparé 2-Aminomethyl-8-methyl-nonanoic acid from 2,6-di-t-butyl-4-methylphenyl cyclopropylcarboxylate. m/z 228.2 (M+). 1.0

W

Example 14. 2-Aminomethyl-4-ethyI-hexanoic acid. A procedure similar to 2-aminomethyl-4,8-dimethyl-nonanoic acid was usedto préparé 2-aminomethyl-4-ethyl-hexanoic acid from 4-ethyl hexanoic acid. m/z174.1. 15

Example 15. 3(S)-Amino-3,5-dimethyI-heptanoic acid. 2-Methyl-propane-2(S)-sulfinic acid (l,3-dimethyl-pentÿlidene)-amide: Asolution of (S)-(-)-2-methyl-2-propanesulfonamide (500 mg, 4.1 mmol), 4-methyl-2-hexanone (470 mg, 4.1 mmol), and Titanium(IV) ethoxide (1.7 mL, 8.3 mmol) was 20 heated at reflux for 18 h. The reaction mixture was poured into 20 mL brine with rapid stirring. The resulting solution was filtered through celite, and the organic layer

I — was separated. The aqueous layer was extracted with ethyl acetate (2x20 mL). Thecombined organics were dried (Na2SO4), filtered, and concentrated. The résultant oilwas purified by silica gel chromatography (25% EtOAc in hexane) to give 575 mg of 25 2-Methyl-propane-2(S)-sulfinic acid (l,3-dimethyl-pentylidene)-amide as a yellowoil. 3,5-DimethyI-3-(2-methyl-propane-2(S)-sulfinylamino)-hepianoic acidmethyl ester: To a-78 °C solution of lithium bis(trimethylsilyl)amide (5.1 ml of a 1 30 M solution in THF) in THF (6 mL) was added methyl acetate ((0.41 mT., 5.1 mmol)dropwise. After stirring for 20 min, a solution of chlorotitanium triisopropoxide (2.5ml, 10 mmol) in THF (3 mL) was added dropwise. After 1 hour, 2-MethyI-propane- 012899 74 2(S)-sulfinic acid (l,3-dimethyl-pentylidene)-amide (560 mg, 2.6 mmol) in THF (3mî.) was added dropwise at -78 °C. The reaction was stiired at -78 °C for 5 h, andthen quenchcd by the addition of 10 mL ammonium chloride solution and warmed toroom température. The mixture was diluted with 10 mL water, and filtered. The 5 aqueous layer was extracted with ethyl acetate (2x20 mL). The combined organicswere washed with brine, dried (NaiSO#), filtered, and concentrated. The résultant oilwas purified by silica gel chromatography (30% EtOAc in hexane) to give 360 mg of 3.5- Dimethyl-3-(2-methyl-propane-2(S)-sulfinylamino)-heptanoic acid methyl ester. 10 3(S)-Àmino-3,5-dimethyl-heptanoicacid: 3,5-Dimethyl-3-(2-methyl- propane-2(S)-sulfînylamino)-heptanoic acid methyl ester (360 mg, 1.2 mmol) wasdissolved in 6 N HCl (2 mL) and dioxane (2 mL) and heated at 100 C for 6 h. Themixture was cooled to room température, diluted with water, and extracted withEtOAc (15 mL). The organics were purified by ion exchange chromatography to give 15 3(S)-Amino-3,5-dimethyl-heptanoic acid (270 mg) and then repurification by silicagel chromatography (70:25:5 C^CL/MeOH/NELOH) to give 203 mg of 3(S)-Amino- 3.5- dimethyI-heptanoic acid as a white solid. m/z 174 (C9H19NO2+H).

Exao oie 16. 3(S)-Amino-3,5-dimethyl-nonanoic acid. 20 A procedure similar to that of 3(S)-Amino-3,5-dimethyl-heptanoic acid was used to préparé 3(S)-Amino-3,5-dimethyl-nonanoic acid, m/z 202.1 (C11H23NO2+H).

Pharmaceutical Composition Examples 25 In the l'ollowing Examples, the term ‘active compound’ or ‘active ingrédient’ refers to a soi table combination or individual element of an alpha-2-delta ligand and aPDEV inhibitor and/or a pharmaceutically acceptable sait or solvaté, according to theprésent invention. 30 i) Tablet compositions 01289 75

The Ibllowing compositions A and B can be prepared by wet granulation ofingrédients (a) to (c) and (a) to (d) with a solution of povidone, followed by additionof the magnésium stéarate and compression.

Composition A ma/tablet ma/tablet (a) Active ingrédient 250 250 tb) Lactose B.P. 210 26 (c) Sodium Starch Glycollate 20 12 ’d) Povidone B.P. 15 9 1» Magnésium Stéarate _5 J. 500 300 15 20

Composition B mg/tablet ma/tablet ta) Active ingrédient 250 250 Φ) Lactose 150 150 c) Avicel PH 101 60 26 (d) Sodium Starch Glycollate 20 12 !» Povidone B.P. 15 9 (0 Magnésium Stéarate _5 _3 500 300 25 Zomnosition C mg/tablet Active ingrédient 100 Lactose 200 Starch 50 30 ’ovidone 5 Magnésium Stéarate _4 359

I 012899 76

The following compositions D and E can be prepared by direct compression ofthe admixed i ngredients. The lactose used in formulation E is of the directcompression type. 5

Composition D mg/tablet

Active ingrédient 250 10 Magnésium Stéarate 4

Pregelatinised Starch NF15 146 400

Composition E mg/tablet 15 Active ingrédient 250

Magnésium Stéarate 5

Lactose 145

Avicel ' 100 500 20 30

Composition F (Controlled release composition') (a) Active ingrédient mg/tablet 500 (b) Hydroxypropylmethylcellulose 112 (Methocel K4M Premium) (c) Lactose B.P. 53 (d) PovidoneB.P.C. ‘ 28 (e) Magnésium Stéarate _7 700 012899

The composition can be prepared by wet granulation of ingrédients (a) to (c) with a solution of povidone, followed by addition of the magnésium stéarate and compression. 77 5 Composition G (Enteric-coated tablef)

Enteric-coated tablets of Composition C can be prepared by coating the tabletswith 25mg/tablet of an enteric polymer such as cellulose acetate phthalate,polyvinylacetate phthalate, hydroxypropylmethyl-cellulose phthalate, or anionic p polymers of methacrylic acid and methacrylic acid methyl ester (Eudragit L). Exceptfor Eudragit L, these polymers should also include 10% (by weight of the quantity ofpolymer used) of a plasticizer to prevent membrane cracking during application or onstorage. Suilabié plasticizers include diethyl phthalate, tributyl citrate and triacetin. 15

Composition H (Enteric-coated controlled release tablef)

Enteric-coated tablets of Composition F can be prepared by coating the tabletswith 5OmgZtablet of an enteric polymer such as cellulose acetate phthalate, 20 polyvinylacetate phthalate, hydroxypropylmethyl- cellulose phthalate, or anionicpolymers of methacrylic acid and methacrylic acid methyl ester (Eudgragit L).Except for Eudgragit L, these polymère should also include 10% (by weight of thequantity of polymer used) of a plasticizer to prevent membrane cracking duringapplication or on storage. Suitable plasticizers include diethyl phthalate, tributyl 25 citrate and triacetin. (ii) Capsule compositions 30

Composition A 012899 78

Capsules can be prepared by admixing the ingrédients of Composition Dabove and filling two-part hard gelatin capsules with the resulting mixture. Composition S finira) mav be prepared in a similar manner. 5 Composition B mg/cansule (a) Active ingrédient 250 (b) Lactose B.P. 143 10 (c) Sodium Starch Glycollate 25 (d) Magnésium Stéarate _2 420 Corn p osition C 15 ma/capsule (a) Active ingrédient 250 (b) Macrogol 4000 BP 350 600 20

Capsules can be prepared by melting theMacrogol 4000 BP, dispersing theactive ingrédient in the melt and filling two-part hard gelatin capsules therewith. 25 Composition D mg/capsule Active ingrédient 250 Lecithin 100 - Arachis Oil 100 30 450

Capsules can be prepared by dispersing the active ingrédient in the lecithinand arachis cil and filling soft, elastic gelatin capsules with the dispersion.

η Π 'ϋ-4 Μ: 79

Composition Ε (Controlled release capsule) mg/capsule (a) Active ingrédient 250 (b) Microcrystalline Cellulose 125 (c) Lactose BP 125 (d) Ethyl Cellulose .13 513 10

The controlled release capsule formulation canbe preparedby extrudingmixed ingrédients (a) to (c) using an extruder, then spheronising and drying theextrudate. The dried pellets are coated with a release controlling membrane (d) andfilled into two-part, hard gelatin capsules. 15

Composition F (Enteric capsule) mg/capsule (à) Active ingrédient 250 (b) Microcrystalline Cellulose 125 (c) Lactose BP 125 (d) Cellulose Acetate Phthalate 50 (e) Diethyl Phthalat _5 555 25 The enteric capsule composition can be prepared by extruding mixed ingrédients (a) to (c) using an extruder, then spheronising and drying the extrudate.The dried pellets are coated with an enteric membrane (d) containing a plasticizer (e)and filled inlo two-part, hard gelatin capsules. 30 Composition G (Enteric-eoated controlled release capsule) 012899 80

Enteric capsules of Composition E can be prepared by coating the controlled-release pellets with 50mg/capsule of an enteric polymer such as cellulose acetatephthalate, polyvinylacetate phthalate, hydiOxypropylmethylcellulose phthalate, oranionic polymers of methacrylic acid and methacrylic acid methyl ester (Eudragit L). 5 Except for Eudragit L, these polymers should also include 10% (by weight of thequantity of polymer used) or a plasticizer to prevent membrane cracking duringapplication or on storage. Suitable plasticizers include diethyl phthalate, tributylcitrate and l riacetin. 10 (iii) Intravenous injection composition

Acli ve ingrédient 0.200g

Stérile, pyrogen-freephosphatebuffer(pH9.0) to 10ml 15 o

The a .rive ingrédient is dissolved in most of the phosphate buffer at 35-40 C,then made up to volume and filtered through a stérile micropore filter into stérile 10ml glass vials (Type 1) which are sealed with stérile closures and overseals. 20 (iv) 1 otramuscular injection composition

Act i ve ingrédient 0.20 g

Ben yl Alcohol 0.10 g 25 Glycofurol 75 1-45 g

Waler for Injection q.s. to 3.00 ml

The active ingrédient is dissolved in the glycofurol. The benzyl alcohol isthen added and dissolved, and water added to 3 ml. The mixture is then filtered 30 through a Sic ile micropore filter and sealed in stérile 3 ml glass vials (Type 1). (v) > vrup composition 012899 0.25g 1.50g 1.00g 0.005g 0.0125ml 5.0ml 81

Active ingrédientSorbi toi SolutionGlycerol 5 Sodium Benzoate

Flavour

Puril'ied Water q.s. to

15

The sodium benzoate is dissolved in a portion of the purified water and thesorbitol solution added. The active ingrédient is added and dissolved. The resultingsolution is mixed with the glycerol and then made up to the required volume with thepurified water. (vi) Suppository composition mg/suppository

Acli e ingrédient 250

Han Fat, BP (Witepsol H15 - Dynamit NoBel) 1770 2020

20 One- l'ifth of the Witepsol H15 is melted in a steam-jacketed pan at 45 C | maximum, fhe active ingrédient is sifted through a 2001m sieve and added to the molten base with mixing, using a Silverson fitted with a cutting head, until a smootho dispersion is achieved. Maintaining the mixture at 45 C, the remaining Witepsol H15is added to lhe suspension which is stirred to ensure a homogenous mix. The entire 25 suspension : · then passed through a 2501m stainless Steel screen and, with continuouso o stimng, al li ved to cool to 40 C. At a température of 38-40 C, 2.02g aliquots of themixture are .'illed into suitable plastic moulds and the suppositories allowed to cool toroom température. 30 (vi i ' °essary composition mg/pessary 012899 82

Active ingrédient (631m) 250 Anhydrous Dextrose 380 Potato Starch 363 Magnésium Stéarate _7 1000

The above ingrédients are mixed directly and pessaries prepared bycompression of the resulting mixture, 10 (viii) Transdermal composition

Active ingrédient 200mg

Alcohol USP 0.1ml

Hydroxyethyl cellulose 15 The active ingrédient and alcohol USP are gelled with hydroxyethyl cellulose

and packed in a transdermal device with a surface area of 10cnA

Claims

012899 83 CLAIMS

1. A combination comprising a synergistic ratio of an alpha-2-deltaligand and a PDEV inhibitor, or a pharmaceutically acceptable sait or solvaté of any 5 thereof.

15

2. A combination according to claim 1, wherein the alpha-2-delta ligandis selected from gabapentin, pregabalin, [(lR,5R,6S)-6-(Axninomethyl)bicyclo[3.2.0]hept-6-yl]acetic acid, 3-(l-Aminometbyl-cyclohexylmethyl)-4H-[l,2,4]oxadiazol-5-one and C-[l-(lH-Tetrazol-5-ylmethyl)-cycloheptylj-methylamine, (3S,4S)-(l-Aminomethyl-3,4-dimethyl-cyclopentyI)-aceticacid, (loc,3a,5a)(3-amino-methyl-bicyc]o[3.2.0]hept-3-yl)-acetic acid, (3S,5R)-

3. A combination according to claim 1 or 2 where the alpha-2-deltaligand is gabapentin, or a pharmaceutically acceptable sait or solvaté thereof. 20

4. A combination according to claim 1 or 2 where the alpha-2-delta ligand is pregabalin, or a phannaceutically acceptable sait or solvaté thereof.

3-Aminomethyl-5-methyl-octanoic acid, (3S,5R)-3-amino-5-methyl-heptanoic acid,(3S,5R)-3-amino-5-methyl-nonanoic acid and (3S,5R)-3-Amino-5-methyl-octanoicacid, or a pharmaceutically acceptable sait or solvaté thereof.

5. A combination according to any one of daims 1-4 wherein the PDEVinhibitor is selected from: 25 5-[2-ethoxy-5-(4-methyl- l-piperazinylsulphonyl)phenyl]- l-methyl-3-n- propyl-l,6-dihydro-7ü-pyrazolo[4,3-d]pyrimidin-7-one (sildenafil); (6R,12aA)-2,3,6,7,12,12a-hexahydro-2-methyl-6-(3,4-methylenedioxyphenyl)-pyrazino[2',l':6,l]pyrido[3,4-i>]indole-l,4-dione (tadalafil, IC-351); * 2-[2-ethoxy-5-(4-ethyl-piperazin-l-yl-l-sulphonyl)-phenyl]-5-methyl-7-30 propyl-3iï-imidazo[5,l-/][l,2,4]triazin-4-one (vardenafil); 5-[2-ethoxy-5-(4-ethylpiperazin-l-ylsulphonyl)pyridin-3-yl]-3-ethyl-2-[2- methbXyethyl]-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one; 012899 5-(5-acetyl-2-butoxy-3-pyridinyl)-3-ethyl-2-(l-ethyl-3-azetidinyl)-2,6- dihydro-7ZZ-pyrazolo[4,3-d]pyrimidin-7-one; and l-{6-ethoxy-5-[3-ethyl-6,7-dihydro-2-(2-methoxyethyl)-7-oxo-2H- pyrazolo[4,3- d]pyrimidin-5-yl]-3-pyridylsulfonyl}-4-ethylpiperazine; 5 or a phannaceutically acceptable sait or solvaté thereof. 84

6. A combination according to any one of daims 1-5 wherein the PDEVinhibitor is sildenafïl, or a phannaceutically acceptable sait or solvaté thereof. 10

7. A combination according to any one of daims 1-5 wherein the PDEV inhibitor is vardenafil, or a phannaceutically acceptable sait or solvaté thereof.

8. A combination according to any one of daims 1-5 wherein the PDEVinhibitor is tadalafil, or a phannaceutically acceptable sait or solvaté thereof. 15

9. A combination as claimed in any one of daims 1-8 for the curative,prophylactic or palliative treatment of pain. 20

10. A combination according to claim 9 where the pain is neuropathie pain.

11. Use of a combination as claimed in any one of daims 1-8 in themanufacture of a médicament for the curative, prophylactic or palliative treatment ofpain. 25

12. Use according to daim 11 where the pain is neuropathie pain. 30

I 012899 85 1

13. A pharmaceutical composition comprising a therapeutically effectiveamount of a combination as claimed in any one of daims 1-8 together with a suitableexcipient or carrier. 5

14. A synergistic combination for human administration comprising an aIpha-2-deita ligand and a PDEV inhibitor, or phaimaceutically acceptable salts orsolvatés thereof, in a w/w combination range which corresponds to a synergisticcombination range of the order of 1:1 to 10:1 parts by weight in the rat model of CCIinduced static allodynia.

15. A synergistic combination for administration to humans comprising analpha-2-delta ligand and a PDEV inhibitor, or pharmaceutically acceptable salts orsolvatés thereof, according to any one of daims 1-8, where the dose ranges of thealpha-2-dclta ligand and PDEV inhibitor correspond to a synergistic dose range of 1- 15 lOmg/kg and 0.1-lmg/kg respectively, in the rat model of CCI induced staticallodynia. 85 pages Pfiaer Inc.

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